Modification of Resins for Their Use in the Separation, Preconcentration and Determination of Metal Ions

MODIFICATION OF RESINS FOR THEIR USE IN THE SEPARATION, PRECONCENTRATION AND DETERMINATION OF METAL IONS

AKIL AHMAD

Under the supervision of Dr. Aminul Islam Department of Chemistry

Submitted in fulfillment of the requirement of the degree of Doctor of Philosophy

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2011

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY AMINUL ISLAM ALIGARH– 202002, INDIA PhD E-mail: [email protected] Assistant Professor Ph: (Off) 91-571-2703515 Analytical Chemistry Mob.: 91- 9358979659

DATE- 18th May, 2011

Certificate

This is to certify that the thesis entitled “Modification of resins for their use in the separation, preconcentration and determination of metal ions ” being submitted by Mr.

Akil Ahmad, for the award of Doctor of Philosophy in Chemistry, is a bonafide research work carried out by him under my supervision. The thesis, to my knowledge, fulfills the requirements for submission for the award of the degree of Doctor of Philosophy in

Chemistry.

Dr. Aminul Islam

Dedicated in the memory of my Abba Acknowledgement

At the outset, I wish to pay my most humble obeisance to the almighty Allah for giving me the wisdom and inspiration to take up this task. It has been Thy guiding hand that has brought this task to a successful completion.

I express my deepest respect and the most sincere gratitude to my esteemed supervisor Dr. Aminul Islam for his scholastic guidance and ungrudged encouragement throughout the course of research work. His systematic and analytical approach blended with constructive criticism and innovative ideas helped me to understand the basics of research and to overcome many experimental hurdles. I am also grateful to him for the freedom he has given to me.

I am thankful to the Chairman, Department of Chemistry, AMU, Aligarh, for providing the necessary research facilities during my research work. Financial assistance from U.G.C., New Delhi is gratefully acknowledged.

My deep sense of gratitude for the guiding hand of my father late Mr. Jamil Ahmad and the humble and loving care of my mother Mrs. Sajada Begum, who allowed me to study in Ph.D cheerfully putting up with inconvenience and difficulties. I am also thankful to my brother Mr. Shakil Ahmad, Mr. Haider Khan, Mr. Shabbir Khan, Mr. Shakib Khan, Fuzail, Sharique, Jahandad, Zeeshan, Tufail, Humail, Tarique, Ahsan, Saheb, Zubi and my affectionate sisters Kehkasha, Seema, Firoza, Enayat and benign support of Shama Nahid and Asma. My neice Shawaiz deserves a special mention for making the task easier for me. I greatly acknowledge the moral and sympathetic support of all my relatives and well wishers.

I must express a very special appreciation to Md Asaduddin Laskar for his constant encouragement through out my ups and downs and often of great help in difficult times. I also thank to my lab colleague Hilal and Noushi being there for me always whenever their help was needed.

I am indebted to my friends particularly Mr. Shadab, Mr. Shahadat, Mr. Ahmad, Mr. Rehan, Mr. Qamar, Mr. Fazil, Mr. Afzal, Mr. Mannan, Mr. Shahab for their support and wishes at every phase of my life and study.

This acknowledgement will be incomplete without the name ‘Dr. Naheed’ for caring and constant encouragement and her loving sons Muaaz and Zaidu.

I am obliged to Prof. Masood Alam (Jamia Milia Islamia, New Delhi) and the Chairman, Department of Botany, AMU for their kind permission to avail the atomic absorption spectrophotometer facility. The help rendered by Mr. Misbah (Instrumentation Centre) and seminar library staff of the department is also duly appreciated.

Akil Ahmad Dedication Certificate Acknowledgement List of publications

CONTENTS List of figures i List of tables iv Chapter 1: Introduction 1.1 Role of geocycle in the introduction of metals into our environment 1 1.2 Significance and characteristics of preconcentration 10 1.3 Solid-phase extraction (SPE) as a preconcentration method 12 1.4 Types of analytical techniques coupled with preconcentration method 26 1.5 Statistical Treatment of Data 35 1.6 Present work and its scope 39 1.7 Merits of the present work 47 REFERENCES 50

Chapter 2: Experimental 2.1 Instrumentation 75 2.2 Reagents and solutions 77 2.3 Pretreatment of samples 79 2.4 Preparation of chelating resin 82 2.5 Characterization of chelating resins 85 2.6 Recommended procedure for sorption studies of metal ions 86 2.7 Procedures for method validation 90 REFERENCES 91

Chapter 3: Characterization of a chelating resin functionalized via azo spacer and its analytical applicability for the determination of trace metal ions in real matrices

3.1 Introduction 93 3.2 Experimental 94 3.3 Results and discussion 96 3.4 Method validation 107 3.5 Applications 108 3.6 Conclusion 110 REFERENCES 111

Chapter 4: Preparation, Characterization of a Novel Chelating Resin Functionalized with o-Hydroxybenzamide and Its Application for Preconcentration of Trace Metal Ions

4.1 Introduction 114 4.2 Experimental 115 4.3 Results and discussion 116 4.4 Method validation 127 4.5 Applications 129 4.6 Conclusions 131 REFERENCES 134

Chapter 5: A newly developed salicylanilide functionalized Amberlite XAD-16 chelating resin for its use in preconcentration and determination of trace metal ions from environmental and biological samples

5.1 Introduction 137 5.2 Experimental 138 5.3 Results and discussion 140 5.4 Analytical figures of merit 153 5.5 Applications 155 5.6 Conclusions 158 REFERENCES 159

Chapter 6: Flame atomic absorption spectrometric determination of trace metal ions in environmental and biological samples after preconcentration on a new chelating resin containing p-Aminobenzene Sulfonic Acid

6.1 Introduction 164 6.2 Experimental 165 6.3 Results and discussion 167 6.4 Analytical figures of merit 181 6.5 Applications 183 6.6 Conclusions 186 REFERENCES 187 LIST OF FIGURES Page No.

Figure 1.1 Interaction of human with the environment 1

Figure 1.2 SPE operation steps 14

Figure 1.3 Interactions occurring at the surface of the solid sorbent 19

Figure 1.4 Atomic absorption spectrometer block diagram 32

Figure 1.5 Typical representation of the breakthrough curve 45 (i.e. concentration of the analyte at the outlet of the SPE system vs. sample volume percolated through the system) Figure 2.1 Diagram depicting a preconcentration system 78

Figure 2.1 Synthesis of chelating resins by incorporating reagents through azo spacer 84

Figure 3.1 Structure of a monomeric unit of AXAD-4 modified with 94 Salicylic acid (SA); ‘a’ is the probable chelating sites

Figure 3.2 FT-IR spectrum of a) AXAD-4-SA and 97 b) AXAD-4-SA saturated with Cu(II)

Figure 3.3 TGA/DTA curves of AXAD-4-SA 97

Figure 3.4 Dependence of sorption capacity on the pH of the solution 98

Figure 3.5 Kinetics of sorption of metal ions on AXAD-4-SA 99

Figure 3.6 Regenerability of AXAD-4-SA for different metals 102

Figure 3.7 Langmuir sorption isotherms depicting the sorption behaviors 105 of metal ions onto AXAD-4-SA

Figure 3.8 Breakthrough curves for sorption of metal ions: C/Co is the 106 concentration ratio of the effluent to influent

Figure 4.1 Structure of a monomeric unit of AXAD-4 modified with 115 o-Hydroxybenzamide (HBAM); ‘a’ is the probable chelating sites

Figure 4.2 FT-IR spectrum of a) AXAD-4-HBAM and 117 b) AXAD-4- HBAM saturated with Cu(II)

Figure 4.3 TGA/DTA curves of AXAD-4-HBAM 118 i

Figure 4.4 Dependence of sorption capacity on the pH of the solution 119

Figure 4.5 Kinetics of sorption of metal ions on AXAD-4- HBAM 120

Figure 4.6 Langmuir sorption isotherms depicting the sorption behaviors 124 of metal ions onto AXAD-4-HBAM

Figure 4.7 Freundlich sorption isotherms depicting the sorption behaviors 125 of metal ions onto AXAD-4-HBAM

Figure 4.8 Breakthrough curves for sorption of metal ions: C/Co is 126 the concentration ratio of the effluent to influent Figure 5.1 Structure of a monomeric unit of AXAD-16 modified with Salicylanilide; 138 (a) and (b) are the probable chelating sites

Figure 5.2 TGA/DTA curves of AXAD-16-SALD 141

Figure 5.3 FT-IR spectrum of a) AXAD-16-SALD and 142 b) AXAD-16-SALD saturated with Cu(II)

Figure 5.4 Dependence of sorption capacity on the pH of the solution 143

Figure 5.5 Kinetics of sorption of metal ions on AXAD-4-SALD 144

Figure 5.6 Influence of temperature on the distribution ratio of metal ions 145

Figure 5.7a, b Pseudo first and second-order kinetic plots for removal of metal ions 147

Figure 5.8 Langmuir sorption isotherms depicting the sorption behaviors 148 of metal ions onto AXAD-16-SALD

Figure 5.9 Breakthrough curves for sorption of metal ions: C/Co is 153 the concentration ratio of the effluent to influent

Figure 6.1 Structure of a monomeric unit of AXAD-16 modified with 165 p-Aminobenzene sulfonic acid; (a) is the probable chelating site and (b) is the hydrophilic and ion exchange group that enhances the hydrophilicity and sorption capacity of the resin

Figure 6.2 TGA/DTA curves of AXAD-16-ABSA 168

Figure 6.3 FT-IR spectrum of a) AXAD-16-ABSA and 169 b) AXAD-16-ABSA saturated with Cu(II) ii

Figure 6.4 Dependence of sorption capacity on the pH of the solution 170

Figure 6.5 Kinetics of sorption of metal ions on AXAD-16-ABSA 171

Figure 6.6 Influence of temperature on the distribution ratio of metal ions 172

Figure 6.7a Pseudo first-order kinetic plots for removal of metal ions 174

Figure 6.7b Pseudo second-order kinetic plots for removal of metal ions 174

Figure 6.8 Langmuir sorption isotherms depicting the sorption behaviors 176 of metal ions onto AXAD-16-ABSA

Figure 6.9 Breakthrough curves for sorption of metal ions: C/Co is 181 the concentration ratio of the effluent to influent

iii

Abstract

The thesis comprises of six chapters. The first chapter (Introduction) starts with a

summary on the geocycle that depicts the different phases of interaction leading ultimately to

the consumption of metal ions by living beings. This is followed by a discussion on the

sources, biochemical mechanism and the effects of toxicity of heavy metals before dwelling

in length about the significance and methodology of solid phase extraction. It gives an

account of the different types of sorbent employed as metal extractors and discusses the

various probable phenomena involved in the retention of the metals ions on to the sorbent.

This chapter also gives a brief account on the methodology of analytical instrumental

techniques employed during the work. It concludes with a brief discussion on statistical

treatment of analytical data.

The following chapter (Experimental) takes into account the different general

experimental procedures followed during the work. Various details that have been covered in

this chapter includes vendor information of the analytical instruments and reagents employed

during the works, the procedures followed for synthesizing different sorbents and methods

employed for sample digestions. This chapter concludes with a brief account on the

procedures followed during method validation.

The rest of the chapters comprise of a brief introduction followed by the results and

discussion for each of the following works. All the chelating resin were prepared by

immobilizing salicylic acid (SA), o-hydroxybenzamide (HBAM), salicylanilide (SALD), p-

aminobenzene sulfonic acid (ABSA) via azo(-N=N-) spacer after nitration (NO2), reduction

(NH2 group) and azotization. All the chemically modified resins were characterized by elemental analysis, TGA and FTIR spectroscopy. FTIR studies of these resins confirmed the incorporation of azo group besides the appearance of the characteristic bands of the ligands 1

immobilized. TGA studies indicated the presence of at least 1.0 molecule of water per repeat

unit of polymer. All the four resins have been used for preconcentration of metal ions such as

Cd(II), Co(II), Cu(II), Ni(II), Pb(II), Mn(II), Cr(III), Fe(III) and Zn(II). The effects of foreign species, namely NaCl, NaNO3, Na2SO4, Na3PO4, KCl, Sodium citrate, Na2C2O4,

CH3COONa, CaCl2, MgCl2, NaK tartrate, humic acid and fulvic acid, that usually coexist in

water and biological samples, on the efficiency of the Amberlite XAD-4 and Amberlite

XAD-16 modified resins for preconcentration of the above mentioned metal ions, were

studied and their tolerance limits, for each sorbent, were determined.

Chemically modified Amberlite XAD-4 with SA

The elemental analysis of AXAD-4-SA gave 63.36%, 4.56%, and 10.12% for C, H and N, respectively. According to thermogravimetric analysis, the resin was found to be

stable up to 200°C with no significant loss of weight other than the loss due to sorbed water

(5.72%). Weight loss of 14.47% up to 382.71°C in TGA was supported by an endothermic

peak in the DTA curve indicate the loss of functional group due to the degradation of SA

reagent in the chelating resin. Its water regain value and hydrogen ion capacity were found to

be 11.75 and 7.15 mmol g-1 respectively. Both batch and column methods were employed to

study the sorption behavior for the metal ions which were subsequently determined by flame

atomic absorption spectrophotometry. The sorption capacity was found to be 245.0, 156.2,

155.0, 145.0, 125.0, 122.5 and 70 µmol g-1 for Cu(II), Cr(III), Zn(II), Cd(II), Mn(II), Ni(II)

and Co(II) respectively with t1/2 less than 15 min. All the metals could be eluted by 5 mL of 4

-1 mol L HCl/HNO3 resulting in high preconcentration factor of 200-360 up to a low

preconcentration limit of 5.5-10 µg L-1. The accuracy and precision of the developed method

was checked by analyzing standard reference materials. The detection limits were found to be

0.42, 0.57, 0.63, 0.77, 0.94, 0.96 and 1.41 µg L-1, respectively. The analytical utility of the

AXAD-4-SA for preconcentration and determination of metal ions was explored by

analyzing river, canal, sewage and tap water by direct as well as standard addition method.

Chemically modified Amberlite XAD-4 with HBAM

Elemental analysis of AXAD-4-HBAM gave C 67.16%, H 5.59%, and N 15.67%

which are in agreement with calculated values for C15H14N3O2·H2O as %C 66.08, %H 5.72

and %N 15.12. According to thermogravimetric analysis, the resin was found to be stable up

to 200°C with no significant loss of weight other than the loss due to sorbed water (6.51%).

Its water regain value and hydrogen ion capacity were found to be 12.93 and 7.68 mmol g-1 respectively. The optimum pH range (with the half-loading time in min, t1/2) for Cu(II),

Cr(III), Ni(II), Co(II), Zn(II) and Pb(II) ions were 2.0-4.0 (5.5), 2.0-4.0 (7.0), 2.0-4.0 (8.0),

4.0-6.0 (9.0), 4.0-6.0 (12.0) and 2.0-4.0 (15.0) respectively. Comparison of breakthrough and

overall capacities of the metals ascertains the high degree of column utilization (>70%). The

breakthrough capacities for Cu(II), Cr(III), Ni(II), Co(II), Zn(II) and Pb(II) ions were found

to be 0.24, 0.18, 0.16, 0.12, 0.09 and 0.08 mmol g-1. High preconcentration factor of 320-400

up to a low preconcentration limit of 5.0–6.3 ng mL-1 has been achieved for almost all the

metals. The detection limit for Cu(II), Cr(III), Ni(II), Co(II), Zn(II) and Pb(II) were found to

be 0.39, 0.49, 0.42, 0.59, 0.71 and 1.10 ng mL-1 respectively. The chelating resin was highly

selective even in the presence of large concentrations of alkali and alkaline earth metals and various matrix components. The AXAD-4-HBAM has been successfully applied for the analysis of natural water, multivitamin formulation, infant milk substitute, hydrogenated oil,

urine and fish.

Chemically modified Amberlite XAD-16 with SALD

The results of elemental analyses of the dried beads of AXAD-16-SALD gave

68.25%, 5.46%, and 11.02% of C, H and N, respectively, which agrees well with the

calculated values, considering the possible stoichiometry of its repeat unit to be

C21H18N3O2.H2O (C, 69.61%, H, 5.52% and N, 11.60%). Thermal analysis indicated that

the synthesized resin was stable up to 180 oC, above which degradation commences. The

water regain value and hydrogen ion capacity were found to be 12.90 and 6.08 mmol g-1 respectively. The optimum pH range for the maximum sorption of Cu(II), Co(II), Ni(II),

Zn(II), Cr(III), Cd(II) and Pb(II) was observed at pH 6.0-9.0, with the half-loading time, t1/2, ranging from 3.5 to 11.0 min. The breakthrough capacities for Cu(II), Co(II), Ni(II), Zn(II),

Cr(III), Cd(II), and Pb(II) were found to be 697.91, 641.83, 629.32, 551.38, 531.72, 249.11 and 125.36 µmol g-1 with the corresponding preconcentration factor of 440, 380, 380, 360,

280, 280 and 260, respectively. The detection limits were found to be 0.56, 0.64, 0.65, 0.70,

0.75, 0.88 and 1.17 µg L-1 respectively. The Student’s t (t-test) values for the analysis of

standard reference materials were found to be less than the critical Student’s t values at 95%

confidence level. Analysis of natural water, mango pulp, mint leaves and fishes were

successfully performed with excellent results.

Chemically modified Amberlite XAD-16 with ABSA

Elemental analysis of AXAD-16-ABSA gave 53.92 %, 4.95 %, 12.65 % and 5.02 of

C, H, N, and S respectively, which agrees well with the calculated values, considering the possible stoichiometry of its repeat unit to be C14H13N3O3S.H2O (C, 55.08 %, H, 4.91 % N,

13.77 % and S, 5.24 %). The presence of 12.65 % of N suggests the incorporation of at least

3.0 mmol of p-Aminobenzene sulfonic acid if we assume that all the functional groups have

been attached through azotisation. In thermogravimetric analysis, the AXAD-16-ABSA resin

shows an earlier weight loss of 6.05 % up to 150 °C. The water regain capacity was found to

be 9.84 mmol g-1. This value reflects the high hydrophilicity of the resin which is satisfactory for column operation. The overall hydrogen ion capacity amounts to 6.56 mmol g-1 of resin, which may be contributed both by the sulfonic and amino groups present within the molecule. The maximum uptake of Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III) and Pb(II) ions were observed in the pH range 4.0-6.0 with the corresponding half-loading time of 6.5,

7.0, 8.0, 9.0, 11.0, 8.5 and 16.5 min. The sorption data followed Langmuir isotherms and pseudo-second order model. Thermodynamic quantities, ΔH and ΔS, based on the variation of distribution coefficient with temperature were also evaluated. The chelating resin was found to tolerate high contents of various naturally occurring alkali and alkaline earth metals, anions and complexing agents in the determination of these metal ions. The maximum preconcentration factors achieved for Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III) and Pb(II) were 360, 300, 300, 290, 180, 180 and 160, respectively with the corresponding preconcentration limit of 5.55, 6.66, 6.66, 6.89, 11.11, 11.11 and 12.50 µg L-1, respectively.

The validity of the method was checked by analyzing standard reference materials and

recoveries of trace metals after spiking. The precision (RSD) for six successive sorption and

elution cycles of 10 µg of each metal ion carried out under optimum conditions was less than

5 %. and offered detection limit of 0.72, 0.89, 1.05, 0.98, 1.17, 0.69 and 1.91 µg L-1 for

Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III) and Pb(II), respectively. The analytical

applications of the method were explored by analyzing of natural water, mango pulp, mint

leaves, and fish.

List of Tables Page No. Table 1.1 Characteristic features of preconcentration 11 Table 1.2 Chelating agents used for modification of Amberlite XAD-4 resin 48 Table 1.3 Chelating agents used for modification of Amberlite XAD-16 resin 49 Table 2.1 Operating parameters set for FAAS for the determination of elements 75 Table 2.2 Composition of standard reference materials 80 Table 3.1 Kinetics and batch capacity of sorption of metal ions on AXAD-4-SA 100 Table 3.2 Elution of metal ions from the AXAD-4-SA 101 Table 3.3 Tolerance limit of foreign species on sorption of metal ions 103 Table 3.4 Langmuir isotherm constants for sorption of metal ions 104 Table 3.5 Preconcentration and breakthrough profiles of metal ions on AXAD-4-SA 107 Table 3.6 Analysis of metal ions in standard reference materials 108 Table 3.7 Determination of metal ions in natural water after preconcentration by 109 AXAD-4-SA column Table 3.8 Determination of metal ions in capsule and food samples 110 Table 4.1 Kinetics and batch capacity of sorption of metal ions on AXAD-4-HBAM 121 Table 4.2 Percent recovery of metal ions from the AXAD-4-HBAM resin using 122 different volume of varying concentration Table 4.3 Tolerance limit of foreign species on sorption of metal ions 123 Table 4.4 Preconcentration and breakthrough profiles of metal ions on AXAD-4-HBAM 127 Table 4.5 Analysis of metal ions in standard reference materials 128 Table 4.6 Determination of metal ions in natural waters collected from various locations 130 after preconcentration by AXAD-4-HBAM column Table 4.7 Determination of metal ions in multi-vitamin capsule, infant milk substitute 130 and hydrogenated oil Table 4.8 Analysis of Common carbs (Cyprinus carpio) and urine for metal content 131 Table 4.9 Comparison of previous published works with present work using 132-133 various chelating resins in terms of different parameter Table 5.1 Kinetics and batch capacity of sorption of metal ions on AXAD-16-SALD 144 Table 5.2 Kinetics parameter for sorption of different metal ions 146 Table 5.3 Langmuir isotherm constants for sorption of metal ions 149 Table 5.4 Tolerance limit of foreign species on sorption of metal ions 151

Table 5.5 Preconcentration and breakthrough profiles of metal ions 152 Table 5.6 Analysis of standard reference materials for metal ion contents 154 Table 5.7 Preconcentration and determination of metal ions in natural waters collected 156 from various locations Table 5.8 Analysis of Common carbs (Cyprinus carpio), mango pulp and mint leaves 157 for metal content Table 6.1 Kinetics and batch capacity of sorption of metal ions on AXAD-16-ABSA 171 Table 6.2 Kinetics parameter for sorption of different metal ions 173 Table 6.3 Langmuir isotherm constants for sorption of metal ions 176 Table 6.4 Tolerance limit of foreign species on sorption of metal ions 179 Table 6.5 Preconcentration and breakthrough profiles of metal ions 180 Table 6.6 Analysis of SRMs, multivitamin tablets and food samples for metal ion 182 contents Table 6.7 Preconcentration and determination of metal ions in natural waters collected 184 from various locations Table 6.8 Analysis of fish, mango pulp and mint leaves for metal content 185

Chapter 1

Introduction

1.1 Role of geocycle in the introduction of metals into our environment

Air, water, earth and life are strongly interconnected as shown in Figure 1.1, which constitute the major segments of the environment- atmosphere, hydrosphere, geosphere and biosphere. Biogeochemical cycles of matter that involve biological, chemical and geological processes and phenomena manifests the strong interaction among living organism and various spheres of the abiotic (nonliving) environment. Water and air are involved in weathering rocks, producing mineral formations and forming climate; which have profound effects on the geosphere and interchange matter and energy with it. Living systems (biosphere) which largely exist on the geosphere in turn have significant effects on it. However, for better or for worse, the environment in which human must live have been affected irreversibly by technology and industrial activities. Metals in the environment may be present in the different states as solid, liquid or gaseous state or in varying forms as individual elements, organic and inorganic compounds. The movement of metals between environmental reservoirs may or may not involve changes of state. Gaseous and particulate metals may be inhaled and solid and liquid (aqueous- phase) metals may be ingested or absorbed, thereby entering the biosphere [1-3].

Figure 1.1 Interaction of human with the environment

Heavy metals are included within the category of environmental toxins: “Materials which can harm the natural environment even at low concentration, through

1 their inherent toxicity and their tendency to accumulate in the food chain and/or have particularly low decomposition rates”. The redistribution of many toxic metals into the environment, caused by the gradual increase in industrial activity, has increased the possibility of human exposure. Among the various toxic elements, heavy metals like cadmium, lead, and mercury are especially prevalent in nature due to their high industrial use. These metals serve no biological function and their presence in tissues reflects contact of the organism with its environment. They are cumulative poison and are toxic even at low dose [4,5]. The indication of their importance relative to other potential hazards is their ranking by the U.S. Agency for Toxic Substances and Disease Registry, which lists all hazards present in the toxic waste sites according to their prevalence and severity of their toxicity. The first, second, third and sixth hazards on the list are heavy metals: lead, mercury, arsenic and cadmium, respectively [6]. The other metal ions that pose potential dangers to human lives include chromium, copper, zinc, nickel, cobalt, iron and manganese. Herein, sources and effects of these metal ions are discussed.

Lead Lead is a highly toxic cumulative poison in humans and animals. Its cumulative poisoning effects are serious hematological damage, anaemia, kidney malfunctioning, brain damage etc. In humans, chronic lead poisoning is manifested by abnormalities such as encephalopathy, nervous irritability, kidney disease, altered heme synthesis and reproductive functions. Such poisoning is associated with low to intermediate levels of chronic exposure to lead. The principle risk to children from lead is interference with the normal development of their brains. A number of studies have found small but significant neuropsychological impairment in young children due to environmental lead absorbed either before or after birth. In particular, lead appears to have deleterious effects on children’s behavior and attentiveness, and possibly also on their IQs. [7]. Environmental contamination of lead is widespread; the main anthropogenic source of this element is burning of leaded gasoline. Lead is used as a construction material for equipment used in sulfuric acid manufacture, petrol refining, halogenation, sulfonation, extraction and condensation. It is used in storage batteries, alloys, solder, ceramics and plastics. It is also used in the manufacture of pigments, tetraethyl lead and other lead compounds, in ammunition, and for atomic radiation and x-ray protection. Lead is used in aircraft manufacture, building construction materials (alloyed with copper, zinc, magnesium, manganese and silicon), insulated cables and wiring, household utensils, laboratory equipment, packaging materials, reflectors, paper industry, printing 2

inks, glass industry, water purification and waterproofing in the textile industry. The primary sources, for low to intermediate levels of chronic exposure to lead, are food, water and air. About 50% of lead is absorbed with inhalation of dusts, 10–15% absorbed orally, out of which 90% is distributed to bones [8]. In natural water its typical concentration lies between 2 and 10 ng mL−1, whereas, the upper limit recommended by WHO is less than 10 ng mL−1 [9].

Copper Excess copper interferes with zinc, a mineral needed to make digestive enzymes [10]. Physical conditions associated with copper imbalance include arthritis, fatigue, adrenal burnout, insomnia, scoliosis, osteoporosis, heart disease, cancer, migraine headaches, seizures, fungal and bacterial infections including yeast infection, gum disease, tooth decay, skin and hair problems and female organ conditions including uterine fibroids, endometriosis and others. Mental and emotional disorders related to copper imbalance include depression, mood swings, fears, anxiety, phobias, panic attacks, violence, autism, schizophrenia, and attention deficit disorder [11]. Copper imbalance in children is associated with delayed development, attention deficit disorder, anti-social and hyperactive behavior, autism, learning difficulties and infections such as ear infections. Copper is primarily used as a metal or an alloy (e.g., brass, bronze, gun metal). Copper sulfate is used as a fungicide, algaecide and herbicide. Copper particulates are released into the atmosphere by windblown dust; volcanic eruptions; and anthropogenic sources, primarily copper smelters and ore processing facilities [12]. Copper particles in the atmosphere will settle out or be removed by precipitation, but can be resuspended into the atmosphere in the form of dust. Copper is released into waterways by natural weathering of soil and rocks, disturbances of soil, or anthropogenic sources (e.g., effluent from sewage treatment plants) [12]. Another source of copper is drinking water that remained in copper water pipes, or copper added to your water supply.

Zinc Zinc is an essential trace element of great importance for humans, plants and animals. It plays an important role in several biochemical processes and its compounds have bactericidal activity. Zinc phosphide, which is used as an active ingredient in rodenticide, reacts with water and acid in the stomach to release phosphine gas which in turn causes cell toxicity with necrosis of the gastrointestinal tract. Oral zinc increases faecal excretion of copper and blocks the absorption of ingested minerals. In this case, a 3 series of complex zinc-copper relationship and metabolism resulted in the inhibition of copper absorption and increased faecal loss of copper through saliva, gastric juices and biliary secretions. Breathing large amounts of zinc (as dust or fumes) can cause a specific short-term disease called metal fume fever. Inhalation of fumes may result in sweet taste, throat dryness, cough, weakness, generalized aching, chills, fever, nausea and vomiting. Zinc chloride fumes have caused injury to mucous membranes and pale gray cyanosis. Ingestion of soluble salts may cause nausea, vomiting and purging [10,13,14]. Zinc has many commercial uses as coating to prevent rust, in dry cell batteries, and mixed with other metals to make alloys like brass and bronze. Zinc compounds are widely used in industry to make paint, rubber, dye, wood preservatives, and ointments [14]. Also used for galvanizing sheet iron; as ingredient of alloys such as bronze, brass, Babbitt metal, German silver, and special alloys for die-casting; as a protective coating for other metals to prevent corrosion, for electrical apparatus, especially dry cell batteries, household utensils, castings, printing plates, building materials, railroad car linings, automotive equipment; as reducer (in form of the powder) in the manufacture of indigo and other vat dyes, for deoxidizing bronze; extracting gold by the cyanide process, purifying fats for soaps; bleaching bone glue; manufacture of sodium hydrosulfite; as reagent in analytical chemistry, e.g. in the Marsh and Gutzeit test for arsenic; as a reducer in the determination of iron. Some zinc is released into the environment by natural processes, but most comes from activities of people like mining, steel production, coal burning, and burning of waste.

Cadmium Cadmium may cause renal injuries and may interfere with the renal regulation of calcium and phosphate balance. It was seen that exposure to abnormal levels of cadmium can result in its accumulation in the renal cortex, which causes a series of adverse subclinical reactions such as hypercalciurium, renal stones and renal tubular dysfunction besides probable development of carcinogenic activity in organisms [15]. The toxicity of cadmium may involve its binding to key cellular sulfhydryl groups, its competition with other metals (zinc and selenium) for inclusion in metalloenzymes, and its competition with calcium for binding sites on regulatory proteins such as calmodulin. The lack of an effective elimination pathway is responsible for cadmium’s biologic half-life of 10-30 years. Chronic effects of cadmium exposure are dose-dependent and include anosmia, yellowing of teeth, emphysema, minor changes in liver function, microcytic hypochromic anemia unresponsive to iron therapy, renal tubular dysfunction characterized by 4

proteinuria and increased excretion of β2-microglobulin and (with prolonged poisoning) osteomalacia leading to bone lesions and pseudo fractures [6]. Cadmium is an industrial waste or by-product, which has a great environmental concern. Cadmium is used in many industrial processes, such as a constituent of easily fusible alloys, soft solder, electroplating and deoxidizer in nickel plating, engraving processes, electrodes for vapor lamps, photoelectric cells, and nickel-cadmium storage batteries [16]. It's wide technological use in fertilizers, mining, pigments, as well as its delivering from oil and coal burning and residues incineration; bring about an extensive anthropogenic contamination of soil, air and water.

Nickel Nickel is a moderately toxic element as compared with other transition metals. However, it is known that inhalation of nickel and its compounds can lead to serious problems, including respiratory system cancer. Moreover, nickel can cause a skin disorder known as nickel-eczema. The most common adverse health effect of nickel in humans is an allergic reaction. People can become sensitive to nickel when things containing it are in direct contact with the skin, when they eat nickel in food, drink it in water, or breathe dust containing it. Less frequently, allergic people have asthma attacks following exposure to nickel. Lung effects, including chronic bronchitis and reduced lung function, have been observed in workers who breathed large amounts of nickel. Headache, dizziness, shortness of breath, vomiting, and nausea are the initial symptoms of overexposure; the delayed effects (10 to 36 h) consist of chest pain, coughing, shortness of breath, bluish discoloration of the skin, and in severe cases, delirium, convulsions, and death. [10]. Long-term exposure can cause decreased body weight, heart and liver damage, and skin irritation. High levels of Ni in the diet may be associated with an increased risk of thyroid problems, cancer, and heart disease [17,18]. Less frequently, allergic people have asthma attacks following exposure to nickel. Lung effects, including chronic bronchitis and reduced lung function, have been observed in workers who breathed large amounts of nickel. Major sources of exposure are: tobacco smoke, auto exhaust, fertilizers, superphosphate, food processing, hydrogenated-fats-oils, industrial waste, stainless steel cookware, testing of nuclear devices, baking powder, combustion of fuel oil, dental work and bridges. Humans are exposed to it through breathing of air or smoking of tobacco containing nickel, eating of food containing nickel, as well as drinking of water

5 contaminated with nickel and handling of coins and touching of other metals containing nickel [19].

Arsenic Once arsenic is in the body, it binds to hemoglobin, plasma proteins, and leukocytes and is redistributed to the liver, kidney, lung, spleen, and intestines. Arsenic produces cellular damage through a variety of mechanisms. Arsenic binds to enzyme sulfhydryl groups and forms a stable ring, which deactivates the enzyme. The process of deactivating the enzyme causes widespread endothelial cell damage, vasodilation, and leakage of plasma. Massive transudation of fluid into the bowel lumen, mucosal vesicle formation, and tissue sloughing may result in large gastrointestinal fluid losses. Arsenic binds to dihydrolipoic acid, a pyruvate dehydrogenase cofactor, blocking the conversion of pyruvate to acetyl coenzyme A and inhibiting gluconeogenesis. Arsenic competes with phosphates for adenosine triphosphate, forming adenosine diphosphate monoarsine, causing the loss of high-energy bonds. In some forms, arsenic is caustic, exerting a direct toxic effect on blood vessels and large organs. Long-term exposure results in nerve damage and may lead to lung, skin, or liver cancer. Once inhaled, arsine gas combines with hemoglobin in RBCs, causing severe hemolysis and anemia. Patients develop hemoglobinuria and hematuria within several hours of exposure. One of the early warning signs of arsenic poisoning is a "pins and needles" sensation in hands and feet. Long-term oral exposure to inorganic arsenic can result in skin changes including darkening of the skin and the appearance of small "corns" or "warts" on the palms, soles, and torso. Arsenite (should be arsenate) (+5) undergoes biomethylation in the liver to the less toxic metabolites methylarsenic acid and dimethylarsenic acid; biomethylation can quickly become saturated, however, and the result is the deposition of increasing doses of inorganic arsenic in soft tissues [20]. Elevated arsenic (As) levels in the environment are attributable to both natural and anthropogenic sources, including geothermal discharges, industrial products and wastes, agricultural pesticides, wood preservatives and mine drainage. It is also used in drugs, war gases and as a homicidal and suicidal weapon. Other uses of arsenic compounds are in alloys, manufacturing of arsenic compounds (arsenic oxides) and certain glass. Copper and lead ores contain small amounts of arsenic. Arsenic is also a major ingredient of Fowler's solution and continues to be found in some folk remedies [14].

Mercury Mercury was recognized as causing neuro-developmental disabilities including dyslexia, attention deficit hyperactivity disorder, intellectual retardation, and autism [21]. Inhalation of very high concentrations causes acute pulmonary edema and interstitial pneumonitis, which may be fatal. In non-fatal cases dyspnea and coughing may persist. Kidney effects may occur at exposure levels lower than those causing central nervous system effects. Also, mercury vapor may cause “Kawasaki” disease, which seems to be immunologically mediated and is similar to Pink disease. Mercury intoxication also causes reproductive effects. Contact dermatitis from mercury amalgam fillings and mercury sensitivity in dental students has been reported. Repeated or prolonged exposure to mercury vapor is highly toxic to the central nervous system [20]. Exposures to high levels of metallic, inorganic, or organic mercury can permanently damage the brain, kidneys, and developing fetus. Embryo toxicity and teratogenicity of organic mercury compounds have been reported in many test systems. Exposure to methyl mercury is worse for young children than for adults, because more of it passes into children’s brains where it interferes with normal development [22-24]. An organic mercury compound, namely methyl mercury, is produced mainly by small organisms in water and soil. Metallic mercury is used to produce chlorine gas and caustic soda and also in thermometers, amalgams (dental fillings), and batteries. Mercury salts are used in skin-lightening creams and as antiseptic creams and ointments. Mercury is used in scientific and electrical equipments, in the electrolytic production of chlorine and sodium hydroxide; and as a catalyst in polyurethane foam production [25]. Inorganic mercury (metallic mercury and inorganic mercury compounds) enters the air from mining ore deposits, burning coal and waste, and from manufacturing plants. It enters the water or soil from natural deposits, disposal of wastes, and the use of mercury-containing fungicides. Breathing of contaminated air or skin contact during use at workplace (dental, health services, chemical, and other industries that use mercury) represents occupational exposures. Inhalation of mercury vapor is the most important route of uptake of elemental mercury [26].

Chromium Epidemiological studies have consistently shown that human exposure to Cr (VI) compounds is associated with a higher incidence of respiratory cancers [27]. Acute toxic effects occur when one breath very high levels of chromium (VI) in air. It can damage and irritate nose, lungs, stomach, and intestines. People who are allergic to chromium 7 may also have asthma attacks after breathing high levels of either chromium (VI) or (III). Long term exposures to high or moderate levels of chromium (VI) causes damage to the nose (bleeding, itching, and sores) and lungs and can increase risk of non-cancer lung diseases. Ingesting very large amounts of chromium can cause stomach upsets and ulcers, convulsions, kidney and liver damage, and even death. Skin contact with liquids or solids containing chromium (VI) may lead to skin ulcers [11,13]. Stainless steel welding, particularly the manual metal arc welding method, may well be the most common source of occupational exposure to Cr(VI), given the fact that there are millions of stainless steel welders worldwide. Chromium is used in manufacturing chrome-steel or chrome-nickel-steel alloys (stainless steel) and other alloys, bricks in furnaces, and dyes and pigments, for greatly increasing resistance and durability of metals and chrome plating, leather tanning, and wood preserving. Handling or breathing sawdust from chromium treated wood, manufacturing, disposal of products or chemicals containing chromium, or fossil fuel burning release chromium to the air, soil, and underground water [28].

Manganese Exposure to atmospheric Mn at high concentration is a risk factor in humans that can manifest as neuronal degeneration resembling Parkinson's disease (PD). Although the underlying mechanism of Mn and dopamine (DA) interaction-induced cell death remains unclear, however, Mn exposure alone to mesencephalic cells for 24h induced minimal apoptotic cell death [29]. Increased manganese intake impairs the activity of copper metallo-enzymes. Excess manganese interferes with the absorption of dietary iron. Long- term exposure to excess levels may result in iron-deficiency anemia. High manganese levels indicate problems with calcium and/or iron metabolism [11,13]. Symptoms of toxicity mimic those of Parkinson's disease (tremors, stiff muscles) and excessive manganese intake can cause hypertension in patients older than 40 [30]. Symptoms of increased manganese levels include psychiatric illnesses, mental confusion, impaired memory, loss of appetite, mask-like facial expression and monotonous voice, spastic gait and neurological problems. Manganese toxicity can cause kidney failure, hallucinations, as well as diseases of the central nervous system. Uses of Mn include: (i) iron and steel production; (ii) manufacture of dry cell batteries; (iii) production of potassium permanganate and other Mn chemicals; (iv) oxidant in the production of hydroquinone; (v) manufacture of glass; (vi) textile bleaching; (vii) oxidizing agent for electrode coating in welding rods; (viii) matches and 8

fireworks; and (ix) tanning of leather [31]. Organic compounds of Mn are present in the fuel additive, methylcyclopentadienyl manganese tricarbonyl (MMT), fungicides (e.g., maneb and mancozeb), and in contrast agents used in magnetic resonance imaging. The primary anthropogenic sources of Mn in ambient air include emission of Mn from industrial sources such as ferroalloy production plants, iron and steel foundries, power plants, and coke ovens and re-entrainment of soils containing Mn [32]. Well water rich in manganese can be the cause of excessive manganese intake and can increase bacterial growth in water. Manganese poisoning has been found among workers in the battery manufacturing industry.

Cobalt Cobalt metal particles, when inhaled in association with other agents such as metallic carbides (hard metals) or diamond dust, may produce an interstitial lung disease termed "hard metal disease" or "cobalt lung". Acute toxicity of cobalt may be observed as effects on the lungs, including asthma, pneumonia and wheezing, that have been found in workers who breathed high levels of cobalt in the air. The International Agency for Research on Cancer (IARC, USA) has determined that cobalt is a possible carcinogen to humans. Studies in animals have shown that cobalt causes cancer when placed directly into the muscle or under the skin [11,13,33]. Vast applications of cobalt in various arrays of products and processes such as its use in alloys, batteries, catalysts, pigments and coloring, make this element to be considered as an important metal in various industries [34]. Cobalt enters the environment from natural sources and from the burning of coal and oil. Workers may be exposed to cobalt in industries that process it or make products containing cobalt [35].

Iron Excessive iron leads to tissue damage as a result of formation of free radicals [36]. Long term over consumption of iron may cause hemosiderosis, a condition characterized by large deposits of the iron storage protein hemosiderin in the liver and other tissues. Iron overload is most often diagnosed when tissue damage occurs, especially in iron- storing organs, such as the liver. Infections are likely to develop because bacteria thrive on iron rich blood. Ironically, some of the signs of iron overload are analogous to those of iron deficiency: fatigue, headache, irritability, and lowered work performance. Other common symptoms of iron overload include enlarged liver, skin pigmentation, lethargy, joint diseases, loss of body hair, amenorrhea, and impotence. Untreated hemochromatosis 9

aggravates the risks of diabetes, liver cancer, heart disease and arthritis. In cases of iron overload the natural storage and transport proteins are overwhelmed and the iron spills over into other tissues and organs, such as the muscle, spleen and liver [37], proving to be toxic [38]. Iron is included in the quality control of industrial and commercial products such as petroleum, alloys, foods, beverages etc [20]. Occasionally, iron pipes may also be a source of iron in water. Water percolating through soil and rock can dissolve minerals containing iron and manganese and hold them in solution.

1.2 Significance and characteristics of preconcentration

Determination of toxic metal ions in environmental samples, wastewater, various natural water bodies and biological fluids is necessary for environment monitoring, assessment of occupational and environmental exposure to toxic metals and its impact on the ecosystem. The low concentrations of the metal ions and the strong interference of matrices present in association with it, in real samples, poses difficulty in its direct determination despite the availability of sophisticated instrumental methods, with excellent sensitivity and multielemental analysis capability [39-46]. A radical way to eliminate matrix effects is a preliminary separation of macro components by a relative, or absolute, preconcentration of trace metals. Preconcentration can be of two types: absolute and relative preconcentration. Absolute prenconcentration involves the transfer of trace elements from a large mass of sample into a small mass, e.g. by evaporation or by solvent extraction into a small volume of an organic phase. Relative preconcentration involves at least partial separation of the components when their concentrations differ very much. Its main aim is often to exchange the matrix for a suitable collector (generally of smaller mass) to prevent its interference in the determination. In some cases, it is difficult to define a boundary between absolute and relative preconcentration. Relative preconcentration increases the mass ratio of trace elements to main components (the solvent is not considered as a major component in this case). Depending on the purpose, trace elements can be concentrated selectively or in groups and either separation of the matrix or separation of the trace components can be used. Removal of the matrix is reasonable if used in combination with multi element 10 determination techniques, e.g. spectrochemical analysis, but only if the matrix is of simple composition. Matrix removal is used especially in analysis of high-purity metals. If the matrix contains several elements forming complex compounds (geological and biological materials), it is better to separate the trace elements. Sometimes, there is no need to remove the matrix completely; the process is then called "enrichment". However, it is usually more profitable to change to another matrix which better meets the demands of the subsequent determination, simplifies a calibration, etc. Several such collectors are suitable for determinations by different techniques. For instance, carbon powder can be analyzed by a spectrographic method or by flameless atomic-absorption spectrophotometry [47]. Some quantitative characteristic features used for description of prenconcentration are listed in Table 1.1

Table 1.1 Characteristic features of preconcentration Recovery (R) R = QT/Q°T, where QT and Q°T are respectively the quantities of trace element in the concentrate and in the sample. It is usually expressed as a percentage.

Concentration coefficient (K) K = (QT/QM) / (Q°T/Q°M), where Q°M and QM are respectively the amounts of matrix before and after

preconcentration. If R = 100% then K = Q°M/QM.

Separation coefficient (S) S = (QM/QT) / (Q°M/Q°T) = 1/K Preconcentration factor Maximum volume of sample / minimum eluent volume that gives quantitative recovery of analyte Preconcentration limit (µg L-1) Minimum concentration up to which preconcentration (maximum volume) is feasible.

Of the analytical techniques for preconcentration and separation, SPE has been preferred over conventional solvent extraction and coprecipitation. The markedly lower quantity of reagents required, the fact that solid phase can be repeatedly used, higher concentration factors, and simplicity in handling and transfer are frequently quoted as advantages [48-51]. Sorption and ion exchange have been studied for different analytical applications [52–60] using various support materials. Many different methods are used for analytical preconcentration [61]. They can be classified according to the nature of the separations (chemical and physical methods) used and the number and nature of the phases involved in the separation process.

 Solvent extraction [62-64]  Co-precipitation [65-67]  Evaporation methods [68]  Electrochemical Techniques [69-70]  Crystallization [71]  Flotation [72]  Membrane filtration [73]  Cloud point extraction [74]  Solid-phase extraction (SPE) [75,76]

1.3 Solid-phase extraction (SPE ) as a preconcentration method

It is a separation process that is used to remove solid or semi-solid compounds from a mixture of impurities based on their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. The separation ability of solid phase extraction is based on the preferential affinity of desired or undesired solutes in a liquid (mobile phase) for a solid (stationary phase) through which the sample is passed. Impurities in the sample are either washed away while the analyte of interest is retained on the stationary phase, or vice-versa. Analytes that are retained on the stationary phase can then be eluted from the solid phase extraction cartridge with the appropriate solvent. The preconcentration methods utilizing solid sorbents are considered to be superior than the liquid-liquid extraction in terms of simplicity, rapidity, and the ability to obtain a high enrichment factor. Particularly, solid phase extraction has been demonstrated in various procedures to be a very effective preconcentration technique in combination with atomic absorption spectrometry. The main advantage of this technique is the possibility of using a relatively simple detection system with flame atomization instead of a flameless technique, which require more expensive equipment and are usually much more sensitive to interferences from macrocomponents of various natural matrices [39].

1.3.1 History and development of SPE The history of using solid phases to isolate drugs from biological samples dates back to 1923 when permutite was used [77] and then subsequently silicic acid [78] for extracting adrenaline. In the 1950s, alumina columns were used [79] for extracting 12

adrenaline and noradrenaline from blood samples. Then successful extraction of catechol bases, adrenaline and histamine from crude extracts of glands, using Amberlite IRC-50 [80] was performed. Later a method was developed to extract catechol amines from tissues using cation exchange resin Dowex 50 [81]. By the mid 1960s more complex samples were being tried. A method using cation exchange paper chromatography [82], which was qualitative in nature, was developed to detect a number of narcotics, tranquilizers, amphetamines and barbiturates from urine samples but the method was more. In the 1970s, the stress was to develop techniques which were more sensitive. Amberlite XAD-2 resin columns were used to quantify narcotic analgesics from urine and could determine as low as 0.6 mg mL of urine [83]. It is noted that, in most of the above cases, the principle of ion exchange was used to separate drugs from biological samples. Subsequently, adsorption phenomenon was tried on charcoal to concentrate a number of drugs (barbiturate, glutathimide, ethchlorvynol, amphetamine, phenothiazine, quinine, morphine, cocaine and its metabolites) from urine and achieved an average detection limit of 1 mg mL-1 of urine [84]. By the late 1970s, HPLC technology had made rapid progress and one of the major developments was the use of silica and bonded silica as the stationary phase. Waters Associates and Analytichem International were among the first to develop the concept of using sorbents, similar to those used in HPLC, packed in miniature columns, to isolate drugs and chemicals from other interfering impurities of test samples. Specifically, small disposable cartridges containing silica, bonded silica and other phases were developed for commercial use and these were called SPE columns. Cl8 SepPak@ columns (Waters Associates) were evaluated for the extraction of tricyclic antidepressants from biological samples [85]. Solid-phase extraction is now emerging as a very important sample preparation technique. It is preferred over other traditional procedures, such as liquid-liquid extraction (LLE), mainly because it is more efficient and much less time-consuming.

1.3.2 Basic principles The principle of SPE is similar to that of liquid-liquid extraction (LLE), involving a partitioning of solutes between two phases. However, instead of two immiscible liquid phases, as in LLE, SPE involves partitioning between a liquid (sample matrix) and a solid (sorbent) phase. This sample treatment technique enables the concentration and purification of analytes from solution by sorption on a solid sorbent. The basic approach involves passing the liquid sample through a column, a cartridge, a tube or a disk containing an adsorbent that retains the analytes. After the 13

entire sample has been passed through the sorbent, retained analytes are subsequently recovered upon elution with an appropriate solvent. The first experimental applications of SPE started fifty years ago [86,87].

1.3.3 Technique An SPE method always consists of three to four successive steps, as illustrated in Figure 1.2.

Figure 1.2 SPE operation steps.

STEP 1: The solid sorbent should be conditioned using an appropriate solvent, followed by the same solvent as the sample solvent. Significance:  This step is crucial, as it enables the wetting of the packing material and the solvation of the functional groups.  In addition, it removes possible impurities initially contained in the sorbent or the packaging.  Also, this step removes the air present in the column and fills the void volume with solvent.  The nature of the conditioning solvent depends on the nature of the solid sorbent.  Care must be taken not to allow the solid sorbent to dry between the conditioning and the sample treatment steps, otherwise the analytes will not be efficiently retained and poor recoveries will be obtained.  If the sorbent dries for more than several minutes, it must be reconditioned.

STEP 2: The second step is the percolation of the sample through the solid sorbent. Depending on the system used, volumes can range from 1 mL to 1000 mL. The 14

sample may be applied to the column by gravity, pumping, aspirated by vacuum or by an automated system.

Significance:  The sample flow-rate through the sorbent should be low enough to enable efficient retention of the analytes, and high enough to avoid excessive duration.  During this step, the analytes are concentrated on the sorbent.  Even though matrix components may also be retained by the solid sorbent, some of them pass through, thus enabling some purification (matrix separation) of the sample.

STEP 3: The third step (which is optional) may be the washing of the solid sorbent with an appropriate solvent, having low elution strength, to eliminate matrix components that have been retained by the solid sorbent, without displacing the analytes. Significance:  A drying step may also be advisable, especially for aqueous matrices, to remove traces of water from the solid sorbent.  This will eliminate the presence of water in the final extract, which, in some cases, may hinder the subsequent concentration of the extract and or the analysis.

STEP 4: The final step consists in the elution of the analytes of interest by an appropriate solvent, without removing retained matrix components. Significance:  The solvent volume should be adjusted so that quantitative recovery of the analyte is achieved with subsequent low dilution.  In addition, the flow-rate should be correctly adjusted to ensure efficient elution.  It is often recommended that the solvent volume be fractionated into two aliquots, and before the elution to let the solvent soak the solid sorbent.

1.3.4 Mechanism of retention Adsorption of trace elements on the solid sorbent is required for preconcentration (Figure 1.3). The mechanism of retention depends on the nature of the sorbent, and may include simple adsorption, chelation or ion-exchange. Active functional groups of the

ligand moiety are responsible for the selective chelation with the metal ions whereas macro porous polymeric support offers large surface area.

Adsorption Trace elements are usually adsorbed on solid phases through van der Waals forces or hydrophobic interaction. Hydrophobic interaction occurs when the solid sorbent is highly non-polar (reversed phase). The most common sorbent of this type is octadecyl- bonded silica (C18-silica). More recently, reversed polymeric phases have appeared, especially the styrene-divinylbenzene copolymer that provides additional pi-pi interaction when p-electrons are present in the analyte [88]. Elution is usually performed with organic solvents, such as methanol or acetonitrile. Such interactions are usually preferred with online systems, as they are not too strong and thus they can be rapidly disrupted. However, because most trace element species are ionic, they will not be retained by such sorbents.

Chelation Several functional group atoms are capable of chelating trace elements. The atoms most frequently used are nitrogen (e.g. N present in amines, azo groups, amides and nitriles), oxygen (e.g. O present in carboxylic, hydroxyl, phenolic, ether, carbonyl, phosphoryl groups) and sulfur (e.g. S present in thiols, thiocarbamates and thioethers). The nature of the functional group will give an idea of the selectivity of the ligand towards trace elements. In practice, inorganic cations may be divided into 3 groups:– 1. Group I-‘hard’ cations: these preferentially react via electrostatic interactions (due to a gain in entropy caused by changes in orientation of hydration water molecules); this group includes alkaline and alkaline-earth metals (Ca2+, Mg2+, Na2+) that form rather weak outer-sphere complexes with only hard oxygen ligands. 2. Group II-‘borderline’ cations: these have an intermediate character; this group contains Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Mn2+. They possess affinity for both hard and soft ligands. 3. Group III-‘soft’ cations: these tend to form covalent bonds. Hence, Cd2+and Hg2+ possess strong affinity for intermediate (N) and soft (S) ligands. For soft metals, the following order of donor atom affinity is observed: O

16 introduce the functional chelating group into the sorbent. For that purpose, three different means are available: 1. The synthesis of new sorbents containing such groups (new sorbents); 2. The chemical bonding of such groups on existing sorbents (functionalized sorbents); and 3. The physical binding of the groups on the sorbent by impregnating the solid matrix with a solution containing the chelating ligand (impregnated, coated or loaded sorbents). The latter remains the most simple to be used in practice. Its main drawback is the possible flush of the chelating agent out of the solid sorbent during sample percolation or elution that reduces the lifetime of the impregnated sorbent. Different ligands immobilized on a variety of solid matrices have been successfully used for the preconcentration, separation and determination of trace metal ions. Chelating agents with a hydrophobic group are retained on hydrophobic sorbents (such as C18-silica). Similarly, ion-exchange resins are treated with chelating agents containing an ion exchange group, such as a sulfonic acid derivative of dithizone (i.e. diphenylthiocarbazone) (DzS), 5-sulfo- 8-quinolinol, 5-sulfosalicylic acid, thiosalicylic acid, chromotropic acid, or carboxyphenylporphyrin (TCPP) [89-92]. Binding of metal ions to the chelate functionality is dependent on several factors:  nature, charge and size of the metal ion;  nature of the donor atoms present in the ligand;  buffering conditions which favor certain metal extraction and binding to active donor or groups; and  nature of the solid support (e.g. degree of cross-linkage for a polymer). In some cases, the behavior of immobilized chelating sorbents towards metal preconcentration may be predicted using the known values of the formation constants of the metals with the investigated chelating agent [93]. However, the presence of the solid sorbent may also have an effect and lead to the formation of a complex with a different stoichiometry than the one observed in a homogeneous reaction [94, 95]. In fact, several characteristics of the sorbent should be taken into account, namely the number of active groups available in the resin phase [93-96], the length of the spacer arm between the resin and the bound ligand [97], and the pore dimensions of the resin [98].

Ion-pairing When a non-polar sorbent is to be used, an ion-pair reagent (IP) can be added to the sorbent [99]. Such reagents contain a nonpolar portion (such as a long aliphatic hydrocarbonated chain) and a polar portion (such as an acid or a base). Typical ion-pair reagents are quaternary ammonium salts and sodium dodecylsulfate (SDS) [100,101]. The non-polar portion interacts with the reversed-phase non-polar sorbent, while the polar portion forms an ion-pair with the ionic species present in the matrix (that could be either free metallic species in solution or complexes).

Ion exchange Ion-exchange sorbents usually contain cationic or anionic functional groups that can exchange the associated counter-ion. Strong and weak sites refer to the fact that strong sites are always present as ion-exchange sites at any pH, while weak sites are only ion-exchange sites at pH values greater or less than the pKa. Strong sites are sulfonic acid groups (cation-exchange) and quaternary amines (anion-exchange), while weak sites consist of carboxylic acid groups (cation-exchange) or primary, secondary and tertiary amines (anion-exchange). These groups can be chemically bound to silica gel or polymers (usually a styrene-divinylbenzene copolymer), the latter allowing a wider pH range. An ion-exchanger may be characterized by its capacity, resulting from the effective number of functional active groups per unit of mass of the material. The theoretical value depends upon the nature of the material and the form of the resin. However, in the column operation mode, the operational capacity is usually lower than the theoretical one, as it depends on several experimental factors, such as flow-rate, temperature, particle size and concentration of the feed solution. As a matter of fact, retention on ion-exchangers depends on the distribution ratio of the ion on the resin, the stability constants of the complexes in solution, the exchange kinetics and the presence of other competing ions. Even though ion-exchangers recover hydrated ions, charged complexes and ions complexed by labile ligands, they are of limited use in practice for preconcentration of trace elements due to their lack of selectivity and their retention of major ions [102]. Yet, for some particular applications they may be a valuable tool. Hence, iron speciation is possible through selective retention of the negative Fe(III)-ferron complex on an anion- exchanger [103]. Selenium speciation is also feasible by selectively eluting Se(IV) and Se(VI) retained on a anion-exchanger [104].

Figure 1.3 Interactions occurring at the surface of the solid sorbent F-functional group; TE-trace element; MS-matrix solvent; MI-matrix ions; ES-elution solvent.

1.3.5 Mechanism of elution of trace elements from the sorbent Similar kinds of interactions (as mentioned in the previous section) usually occur during the elution step. The type of solvent must be correctly chosen to ensure stronger affinity of the trace element to the solvent, to ensure disruption of its interaction with the sorbent (Figure 1.3). Thus, if retention on the sorbent is due to chelation, the solvent could contain a chelating reagent that rapidly forms a stronger complex with the trace metal. Elution may also be achieved using an acid that will disrupt the chelate and displace the free trace element. Similarly, if retention is due to ion exchange, its pH dependence enables the use of eluents with different pH to be used, such as acids. Of prime importance is to selectively elute only the target species. So, if they are more strongly retained on the sorbent than the interfering compounds, a washing step with a solvent of moderate elution strength is highly advisable before elution of the target species with the appropriate solvent.

1.3.6 Selection of Solid Sorbents The nature and properties of the sorbent are of prime importance for effective retention of metallic species. In practice, the main requirements for a solid sorbent are:

 The possibility to extract a large number of trace elements over a wide pH range (along with selectivity towards major ions);  Kinetically faster quantitative sorption and elution;  A high capacity;  Regenerability; and  Accessibility Solid sorbents may be reversed-phase sorbents or normal-phase sorbents. Reversed-phase sorbents usually refer to the packing materials that are more hydrophobic than the sample, which are frequently used with aqueous samples. When hydrophobic supports are used, retention of ionic metal species will require the formation of hydrophobic complexes. This can be achieved through addition of the proper reagent to the sample or through immobilization of the reagent on the hydrophobic solid sorbent. Normal-phase sorbents refer to materials more polar than the sample and they are used when the sample is an organic solvent containing the target compounds.

Nascent polymeric resins as sorbent Nascent styrene–DVB resins such as Amberlite XAD-1180 [105,106], XAD-4 [107], and XAD-16 [108-112] are used directly for enrichment of inorganic species as their halide or thiocyanate complex. The type and quantity of sorbent, hydrophobicity, ionizability of the analytes, sample volume and pH interactively determine the breakthrough volume. Using a styrene–divinylbenzene sorbent, the primary interaction mechanism is via Vander Waals forces; therefore, the more hydrophobic the compound the larger the breakthrough volume will be and the larger the sample size from which quantitative recovery can be expected. This observation can be generalized to other sorbents by stating that regardless of the primary interaction mechanism between the analyte and the sorbent, it holds true that the stronger the interaction, the larger the breakthrough volume will be.

Modification of nascent polymeric resins . Macroporous hydrophobic resins of the Amberlite XAD series [polystyrenedivinylbenzene (PS-DVB) resins] are good supports for developing chelating matrices. Amberlite XAD resins, as the copolymer backbone for the immobilization of chelating ligands, have some physical superiority, such as porosity, uniform pore size distribution, high surface area and chemical stability towards acids, bases, and oxidizing agents, as compared to other resins. In addition to the hydrophobic interaction that also 20

occurs with C18-silica, such sorbents also allow π-π interactions. Due to the hydrophobic character of PS-DVB, retention of trace elements on such sorbents requires the addition of a ligand to the sample. The use of surface-modified PS–DVB copolymers with different polar substituent overcomes the following disadvantages suffered by standard silica-based material used for SPE:  lack of pH stability under acidic or basic conditions,  low breakthrough for polar analytes,  they are not wettable by water alone and always need a conditioning step with a wetting solvent, such as methanol. However, in practice, the resins prepared by impregnation of the ligand are difficult to reuse, due to partial leaching of the ligand (thus resulting in poor repeatability). To overcome this problem, the resin may be chemically functionalized. Chemical modification of PS–DVB copolymers have been carried out by immobilizing varying substituents through different bridging groups.

Amberlite XAD-2 Surface modification: Amberlite XAD-2 resin modified by surface adsorption with oxime [113,114] 1-(2-thiazolylazo)-2-naphthol [113,115], pyrocatechol violet [113], 4-(2- pyridylazo) resorcinol [114], eriochrome blue black R [114], ammonium pyrollidine dithiocarbamate (APDC) [116], tropolone [117], 1-(2-pyridylazo)- 2-naphthol (PAN) [118], 2-(2-thiazolylazo)-p-cresol [119], calmagite [120,121], and organophosphinic acid [122] were used as solid phase extractant sorbents in off-line or online column preconcentration modes.

Chemical modification: Singh et al. chemically immobilized AmberliteXAD-2 with alizarin Red S [123], tiron [124], catechol [125], thiosalicylic acid [126], o-aminophenol [127], chromotropic acid [128], catechol violet [129], salicylic acid [130], and pyrogallol [125] via azo (-N=N-) spacer. A similar synthetic scheme was employed by Jain et al. [131, 132] to chemically immobilize o-vanilline thiosemicarbazone on to Amberlite XAD-2 resin. Dogutan et al. [133] synthesized palmitoyol-8-hydroxyquinoline functionalized Amberlite XAD-2 by a modified procedure described by Suebert et al. [134] through chloromethylation. In 1992, Trojanowicz group [135] chemically immobilized Eriochrome blue black R onto Amberlite XAD-2. Yuan and Shuttler [136] immobilized quinoline-8-ol onto Amberlite XAD-2 and controlled pore glass, and

reported higher enrichment factors with the former as it gave higher flow rates during quantitation of aluminum by FIA-ETAAS.

Amberlite XAD-4 Surface modification: Surface adsorption of Amberlite XAD-4 resin beads with oxine [137-140], APDC [137, 138], 2[2-(5-chloropyridyl)azo]- 5-dimethyl amino phenol (5- ClDMPAP) [139,140], butane–2,3-dionebis(N-pyridinoacetylhydrazone)[141], 2-(5- bromo-2-pyridylazo)-5-diethyl aminophenol (5-Br PADAP) [142-144], 5-phenyl azo-8- quinolinol [145], 1-nitroso-2-napthol [146], and N-benzoylphenylhydroxylamine [147] were used as solid-phase extractants for the trace determination of inorganics using a variety of detection techniques which include spectral and X-ray techniques.

Chemical modification: Azotization was used for immobilzation of o-aminobenzoic acid onto Amberlite XAD-4 resin by Cekic et al. [148]. Jain et al. [149] employed similar synthetic scheme for functionalizing Amberlite XAD-4 with o-vanilline-semicarbazone. Amberlite XAD-4 was functionalized with N-hydroxy ethyl ethylene diamine via acylation by Hirata et al. [150] as per the synthesis procedure described by Dev and Rao [151]. Acid chloride was grafted onto Amberlite XAD-4 [152], Yakin and Apak [153] immobilized maleic acid by electrophilic substitution of the Amberlite XAD-4 resin with maleic anhydride by a Friedel-Crafts reaction. Quinoline-8-ol functionalized Amberlite XAD-4 resin was synthesized by Gladis and Rao [154] through acetylation.

Amberlite XAD-16 Surface modification: Traces of inorganics were enriched on Amberlite XAD-16 resin beads after surface adsorption with a variety of chelates, namely PAN [155], NaDDTC [156,157], 4-(2-thiazoylazo) resorcinol [158,159], N,N-dibutyl-N-benzoylthiourea (DBBT) [160], and di-(2-ethylhexyl phosphoric acid (D2EHPA) [161].

Chemical modification: Azotization was employed to functionalize (bis-2,3,4-trihydroxy benzyl)ethylene diamine(BTBED) [162], 2-{[1-(3,4-Dihydroxyphenyl)methylidene] amino}-benzoic acid (DMABA) [163], 4-{[(2-Hydroxyphenyl)imino]methyl}-1,2- benzenediol (HIMB) [164] and Nitrosonaphthol [165] on to Amberlite XAD-16. D. Prabhakaran et. al. immobilized 1,3-dimethyl-3-aminopropan-1-ol onto Amberlite XAD- 16 via simple condensation mechanism [166].

Apart from this a number of different solid sorbents have been investigated for the preconcentration of trace metals from an aqueous solution. They include:

 Silica gel (Inorganic based sorbents) [167-172]  C-bonded silica gel (Inorganic based sorbents) [173-178]  Other inorganic oxides (Inorganic based sorbents)[179-182]  Divinylbenzene-vinylpyrrolidone copolymers (polymeric organic sorbents)[183- 184]  Polyacrylate polymers (polymeric organic sorbents)[185-187]  Polyurethane polymers (polymeric organic sorbents)[188-192]  Polyethylene polymers (polymeric organic sorbents)[193]  Polytetrafluoroethylene polymers (polymeric organic sorbents)[194-196]  Polyamide polymers (polymeric organic sorbents)[197]  Iminodiacetate-type chelating resins (polymeric organic sorbents)[198-201]  Propylenediaminetetraacetate-type chelating resins (polymeric organic sorbents)[202]  Polyacrylonitrile based resins (polymeric organic sorbents)[203-206]  Ring-opening metathesis polymerization based polymers (polymeric organic sorbents)[207,208]  Carbon sorbents (non-polymeric organic sorbents)[209-215]  Cellulose (non-polymeric organic sorbents)[216-218]  Naphthalene based sorbents (non-polymeric organic sorbents)[219-226]  Molecularly Imprinted Polymers (MIP) [227,228]

1.3.7 Advantages of SPE Reduced detection limit This step is used if the detection limit of the analytical technique is higher than the concentration of trace elements in the sample. Concentration often involves separation of the matrix or the bulk of it, and sometimes a number of interfering minor constituents as well. In a prepared concentrate, the relative concentration of trace elements is usually higher than in the initial sample. Moreover, the possibility of increasing the amount of sample analyzed means that the absolute amounts of elements to be determined can also be increased. As a result, it is possible to reduce the detection limit of trace elements (sometimes very significantly; by a factor of 100 or 1000). This is the main but not the only reason for the widespread use of preconcentration [229]. 23

Preparation of a representative sample Preconcentration is almost essential if trace elements are non-homogeneously distributed in the material. In this case, a representative sample must be quite large; it is difficult to analyze it directly, especially if the method of determination needs a small sample as, for instance, spark-source mass-spectrometry or spectrochemical analysis. In many other cases, preconcentration with preliminary dissolution and production of a small volume of concentrate facilitates the preparation of a representative sample. Samples can be homogenized during other operations also. SPE enjoys superiority over solvent extraction as it is free from difficult phase separation, which is caused by the mutual solubility between water and organic solvent layers [230].

Facilitates calibration Concentration facilitates calibration, especially if there is a lack of standard reference materials. It makes it possible to obtain concentrates with identical matrices in analysis of quite different materials, for example, concentrates on carbon powder in spectrochemical analysis. Reference samples are prepared as concentrates of the same type. There is then no necessity to have standard reference materials for all substances analyzed. Preconcentration with exhaustive removal of the matrix is desirable in the analysis of toxic, radioactive or, if the matrix can be recovered, very expensive materials. Moreover, it is convenient to add elements as internal standards, if necessary, during decomposition of the sample and concentration. Sometimes, preconcentration allows an increase in the number of trace elements which can be determined by a selected technique or makes it possible for the determination technique to be used at all. These advantages of preconcentration make it an important part of trace analysis. In spite of the progress in sensitive instrumental methods of direct analysis, the significance of concentration does not diminish. On the contrary, its possibilities increase, particularly because of new combinations with methods of determination [39,231].

Other attractive features This technique is attractive as it reduces consumption of and exposure to solvents, their disposal costs and extraction time [232]. It also allows the achievement of high recoveries, along with possible elevated enrichment factors. However, different results between synthetic and real samples may be observed [233]; recoveries should be estimated in both cases as far as possible. Its application for preconcentration of trace metals from different samples is also very convenient due to sorption of target species on 24

the solid surface in a more stable chemical form than in solution. Use of carcinogenic organic solvents is avoided and thus the technique is ecofriendly to nature. Finally, SPE affords a broader range of applications due to the large choice of solid sorbents.

High preconcentration factor The use of SPE enables the simultaneous preconcentration of trace elements, removal of interferences, and reduces the usage of organic solvents that are often toxic and may cause contamination. Upon elution of the retained compounds by a volume smaller than the sample volume, concentration of the extract can be easily achieved. Hence, concentration factors of up to 1000 may be attained [229].

Preservation and storage of the species SPE allows on-site pre-treatment, followed by simple storage and transportation of the pre-treated samples with stability of the retained metallic species for several days [234,235]. This point is crucial for the determination of trace elements, as the transport of the sample to the laboratory and its storage until analysis may induce problems, especially changes in the speciation. In addition, the space occupied by the solid sorbents is minimal and avoids storage of bulky containers and the manpower required to handle them.

Selective extraction SPE offers the opportunity of selectively extracting and preconcentrating only the trace elements of interest, thereby avoiding the presence of major ions. This is crucial in some cases, such as with spectrophotometric detection, since the determination of heavy metals in surface waters may necessitate the removal of non-toxic metals, such as Fe or Zn, when they occur at high concentrations [236]. It may also be possible to selectively retain some particular species of a metal, thereby enabling speciation. For example, salen I modified C18-silica is quite selective towards Cu(II) [237], while chemical binding of formylsalicylic acid on amino-silica gel affords selectivity towards Fe(III) [238]. This high selectivity may also be used to remove substances present in the sample that may hinder metal determination, such as lipid substances in the case of biological samples [239]. The chelating resin method is an economical method since it uses only a small amount of ligand and extraction solvent and this also increases the sensitivity of the system.

Automation and possible on-line coupling to analysis techniques SPE can be easily automated, and several commercially available systems have been recently reviewed [239]. In addition, SPE can be coupled online to analysis techniques. On-line procedures avoid sample manipulation between preconcentration and analysis steps, so that analyte losses and risk of contamination are minimized, allowing higher reproducibility [240]. In addition, all the sample volume is further analyzed, which enables smaller sample volume to be used. However, in the case of complex samples, off- line SPE should be preferred due to its greater flexibility, and the opportunity to analyze the same extract using various techniques.

1.3.8 Challenges Some of the challenges of preconcentration are as follows:  Preconcentration increases the analysis time and complicates the analysis for large volume. However, this can be minimized with online systems using flow injection (FI) techniques because of their potential for automation, minimization of reagent and/or sample consumption, and reduced risk of contamination. It ffers all essential prerequisites for trace analysis [241].  It may also lead to losses or contamination of trace elements to be determined.  Special working procedures, reagents of high purity, specially equipped laboratories and special materials for equipments are necessary.

1.3.9 Common SPE applications The common SPE application includes:  Metal ions in various complex real samples,  Pharmaceutical compounds and metabolites in biological fluids,  Drugs of abuse in biological fluids,  Environmental pollutants in drinking and waste water,  Pesticides and antibiotics in food/agricultural matrices,  Desalting of proteins and peptides,  Fractionation of lipids, and  Water and fat soluble vitamins

1.4 Types of analytical techniques coupled with preconcentration method

Some frequently used analytical techniques in combinations with preconcentration may be described as follows: 1.4.1 Adsorptive stripping Voltammetry In trace analysis, mainly of heavy metal ions, Anodic Stripping Voltammetry (ASV) is popular because of the low limit of determination – ranging to sub ppb concentrations, its accuracy and precision, as well as the low cost of instrumentation for this analytical method. ASV is based on previous electrolytical accumulation of the compound to be determined on the working electrode, followed by voltammetric dissolution (oxidation) of the reduced substance formed. In addition, some anions or organic compounds can be accumulated on a mercury electrode to form an insoluble compound with the mercury ions obtained by dissolution of the mercury electrode at positive potentials. In this type of cathodic stripping voltammetry (CSV), the reduction process of the mercury compound on the electrode surface is studied. The most important step, leading to a substantial increase in the sensitivity in both types of methods is electrolytic accumulation of the species on the working electrode. The high sensitivity of adsorptive stripping method is obviously their greatest advantage. On the other hand, a serious drawback is interference from other surface- active substances that may be present in the solution. In this case, competitive adsorption usually occurs and leads to a decrease in the measured current or, at very high surface- active substance (s.a.s.) concentrations, to significant suppression of the signal. Interfering effects depend on the nature of both the analyzed and interfering substances and on their concentration ratio in the determination. Evidently, the interfering effect of s.a.s. can be minimized by employing short accumulation times; however, this approach is not suitable in the determination of trace amounts of analyte. It is then necessary to employ suitable separation of interfering compounds, e.g. the application of LC or gel chromatography and extraction procedures [242-244].

1.4.2 X –Ray fluorescence analysis For the analysis of liquid samples by X-ray fluorescence (XRF) spectrometry, the liquid in a specially designed liquid sample holder is irradiated directly with X-rays. However, for direct analysis of liquid samples, the abundance of effective amounts of analytes in the irradiated volume is insufficient for the determination of trace metals in natural water. Therefore, for the quantitation of trace metals in aqueous samples by XRF,

it is necessary to preconcentrate the analytes through accepted sample pretreatments [245,246]. In this case, group concentratration, and more rarely individual concentration, is used and the concentrate should preferably be a solid which can be analyzed directly. Otherwise, several operations must be successively performed. Thus for example, extracts are often decomposed and the trace elements sorbed on cellulose powder or silica gel carrying functional groups. It is more convenient to do the extraction with low-melting reagents at increased temperature. In this case, the solidified concentrate may at once be pressed into a tablet and analyzed.

1.4.3 Spectrophotometric determination Combinations of concentration, especially extractive, with spectrophotometry in the visible and ultraviolet regions are widely known. Individual concentration steps or successive separations of several elements are usually used. The matrix is very rarely separated in this case. Most frequently, the reagent used for concentration also gives a colored complex with the element to be determined. However, two reagents may also be used: first, the most selective reagent is used for the separation and then, for the determination, a reagent which may not be selective but is the most suitable form for the photometry is employed. The second reagent may also be added after back-extraction or mineralization of the extract [248-250].

1.4.4 Neutron-activation analysis There are two distinct ways of trace concentration in this method: before irradiation and after it. Separation of the matrix before irradiation is necessary if it is strongly activated and the radioisotopes formed have long half-lives. In the concentration, trace components which are strongly activated (but are not to be determined) may also be separated. However, concentration before irradiation nullifies one of the main advantages of activation analysis-that a blank correction is unnecessary. This advantage is still valid, however, in the case of concentration after irradiation. In analytical practice, both variants are used but the second is used more frequently [251-253].

1.4.5 Electrothermal Atomic Absorption Spectrometry Nowadays, electrothermal atomic absorption spectrometry (ETAAS) is most powerful and popular analytical tools for the determination of low concentration of metal ions present in environmental samples and biological materials due to their high 28

selectivity and sensitivity for analyte determination. Nevertheless, they are potentially prone to spectroscopic and/or non-spectroscopic interferences. Various schemes have been suggested to alleviate the interfering effects and facilitate reliable analysis such as protocols ranging from instrument modifications (e.g. background correction) to experimental designs (e.g. standard addition or internal standardization). However, instead of implementing such approaches, there is a much simpler and effective solution to the problem, namely to subject the sample to appropriate pretreatments before it is presented to the detector. Preconcentration addresses these serious problems for the determination of metal ions. Concentration of the desired trace elements can extend the detection limits, remove interfering constituents, and improve the precision and accuracy of the analytical results [254-256].

1.4.6 Inductively coupled plasma optical emission spectrometry (ICP-OES) Inductively coupled plasma optical emission spectrometry is an analytical technique often employed to determine metal ions in various types of samples [257]. But, their sensitivity and selectivity are usually insufficient for direct determination of these contaminants at a very low concentration level in complex matrix environmental samples. Moreover, several types of spectral interference have been reported in the determination of metal ions by ICP-OES. Thus, preconcentration and separation procedures have been devised to allow trace amounts of metal ions to be determined in complex matrices using ICP-OES [258,259].

1.4.7 Inductively coupled plasma mass spectrometry (ICP-MS) Although inductively coupled plasma-mass spectrometry (ICP-MS) capable of the rapid simultaneous analysis of multiple elements over a large range of concentrations from a single aliquot of sample, the high salinity concentrations of open ocean samples can cause substantial salt precipitation and build-up, unpredictable suppression or enhancement effects as well as mask the analyte signal through the formation of isobaric and polyatomic interferences due to the presence of high salt content in the samples. In particular, the matrix elements in the sample can combine with carbon in the atmosphere and/or argon in the plasma and result in the formation of polyatomic species which may interfere with the determination of numerous analytes including transition metals and rare earth elements. In addition, when the sample contains a very high concentration of dissolved salts, e.g. seawater; clogging of the sample introduction system or of the injector tube of the torch may occur. Therefore, direct sample injection is impractical, 29

requiring sample pre-treatment to remove the high salinity matrix by either dilution of the sample or using analyte extraction/separation techniques. Despite the sensitivity of ICP- MS, open ocean trace metal concentrations are often at or below the detection limit, prohibiting the further dilution of samples. It is therefore advisable to concentrate and separate the trace metals from the seawater matrix prior to ICP-MS analysis [260,261].

1.4.8 Inductively coupled plasma atomic emission spectrometry (ICP-AES) Inductively coupled plasma atomic emission spectrometry (ICP-AES) is widely recognized as a multi-element technique for the determination of elemental species, though direct determinations in environmental and biological samples at trace level is difficult, because the aspiration of solutions with high salt concentrations in the plasma can cause problems such as blockage of the nebulizer, considerable background emission, and transport and chemical interferences with a consequent drop in sensitivity and precision. This limitation can be overcome by using enrichment methods in that metals ions of interest from solutions are selectively separated and preconcentrated into smaller volumes to achieve better detection by ICP-AES [262,263].

1.4.9 Atomic absorption spectrometry Flame atomic absorption is a very common technique for detecting metals and metalloids in environmental samples [264]. It is a well established technique for the quantification of nearly 70 elements in a variety of sample types with sensitivity at the ppm level or less. Aqueous samples can be determined with no sample preparation; solid samples must be dissolved or digested. This sample preparation for solids can be time consuming. Although atomic absorption spectroscopy dates to the nineteenth century, the modern form was largely developed during the 1955s by a team of Australian chemists. They were led by Alan Walsh and worked at the CSIRO (Commonwealth Science and Industry Research Organization) Division of Chemical Physics in Melbourne, Australia. The time since 1955 can be divided into seven years period. The first was an induction period (1955-1961) when atomic absorption received attention from only a very few people. This was followed by a growth period (1962-1969) when most of what we see today was developed, and then by a period of relative stability (1969-1976) when atomic absorption contributed greatly to other fields. We are now in a period of great change, which started in about 1876, due to the impact of computer technology on individual laboratory instruments [265]. Flame atomic absorption spectrometry is among the most widely used methods for the determination of the heavy metals at trace levels. This 30 technique presents desirable characteristics such as operational facilities, good selectivity and low cost. However, in the presence of very high excess of diverse ions compared with the level of analyte, some limitations, mainly those related to the sensitivity are observed. In trace analysis, therefore, a preconcentration and/or separation of trace elements from the matrix are frequently necessary to improve the detection limit and selectivity of their determination [39,266]. Particularly, solid phase extraction has been demonstrated in various procedures to be a very effective preconcentration technique in combination with atomic absorption spectrometry. The main advantage of this technique is the possibility of using a relatively simple detection system with flame atomization instead of a flameless technique, which require more expensive equipment and are usually much more sensitive to interferences from macro components of various natural matrices [39].

Principles The technique (Figure 1.4) makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It relies therefore heavily on Beer-Lambert law. In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals for a short amount of time by absorbing a set quantity of energy (i.e. light of a given wavelength). This amount of energy (or wavelength) is specific to a particular electron transition in a particular element, and in general, each wavelength corresponds to only one element. This gives the technique its elemental selectivity. As the quantity of energy (the power) put into the flame is known, and the quantity remaining at the other side (at the detector) can be measured, it is possible, from Beer-Lambert law, to calculate how many of these transitions took place, and thus get a signal that is proportional to the concentration of the element being measured. In order to analyze a sample for its atomic constituents, it has to be atomized. The sample should then be illuminated by light. The light transmitted is finally measured by a detector. In order to reduce the effect of emission from the atomizer (e.g. the black body radiation) or the environment, a spectrometer is normally used between the atomizer and the detector.

Types of Atomizer The technique typically makes use of a flame to atomize the sample [267], but other atomizers such as a graphite furnace [268] or plasmas, primarily inductively coupled plasma, are also used [269].

Figure 1.4 Atomic absorption spectrometer block diagram

When a flame is used it is laterally long (usually 10 cm) and not deep. The height of the flame above the burner head can be controlled by adjusting the flow of the fuel mixture. A beam of light passes through this flame at its longest axis (the lateral axis) and hits a detector.

Analysis of liquids A liquid sample is normally turned into an atomic gas in three steps: 1. Desolvation (Drying) – the liquid solvent is evaporated, and the dry sample remains. 2. Vaporization (Ashing) – the solid sample vaporises to a gas. 3. Atomization – the compounds making up the sample are broken into free atoms.

Radiation Sources The radiation source chosen has a spectral width narrower than that of the atomic transitions.

Hollow cathode lamps Hollow cathode lamps are the most common radiation source in atomic absorption spectroscopy. Inside the lamp, filled with argon or neon gas, is a cylindrical metal cathode containing the metal for excitation, and an anode. When a high voltage is applied across the anode and cathode, gas particles are ionized. As voltage is increased, gaseous ions acquire enough energy to eject metal atoms from the cathode. Some of these atoms

are in excited states and emit light with the frequency characteristic to the metal [270]. Many modern hollow cathode lamps are selective for several metals.

Diode lasers Atomic absorption spectroscopy can also be performed by lasers, primarily diode lasers because of their good properties for laser absorption spectrometry [271]. The technique is then either referred to as diode laser atomic absorption spectrometry (DLAAS or DLAS) [272], or since wavelength modulation most often is employed, wavelength modulation absorption spectrometry.

Interferences Various factors which may interfere with the determination of metal ions are as follows:

 Chemical interferences may also be eliminated by separating the metal from the interfering material. Although complexing agents are employed primarily to increase the sensitivity of the analysis, they may also be used to eliminate or reduce interferences.  The presence of high dissolved solids in the sample may result in interference from non-atomic absorbance such as light scattering. If background correction is not available, a non-absorbing wavelength should be checked. Preferably, samples containing high solids should be extracted.  Ionization interferences occur when the flame temperature is sufficiently high to generate the removal of an electron from a neutral atom, giving a positively charged ion. This type of interference can generally be controlled by the addition, to both standard and sample solutions, of a large excess (1,000 mg/L) of an easily ionized element such as K, Na, Li or Cs.  Spectral interference can occur when an absorbing wavelength of an element present in the sample but not being determined falls within the width of the absorption line of the element of interest. The results of the determination will then be erroneously high, due to the contribution of the interfering element to the atomic absorption signal.  Interference can also occur when resonant energy from another element in a multielement lamp, or from a metal impurity in the lamp cathode, falls within the band-pass of the slit setting when that other metal is present in the sample. This type of interference may sometimes be reduced by narrowing the slit width. 33

 The interference, known as background absorption, arises from the presence in the flame of gaseous molecules, molecular fragments and some time smoke. In addition background effects can be caused by light scatter.

Procedures for reduction of interferences  Ensure if possible that standard and sample solutions are of similar bulk composition to eliminate matrix effects.  Alteration of flame composition or of flame temperature can be used to reduce the likelihood of stable compound formation within the flame.  Selection of an alternative resonance line will overcome spectral interferences from other atom or molecules and from molecular fragments.  Separation, for example by solvent extraction or an ion exchange process, may occasionally be necessary to remove an interfering element.

Background Correction methods The narrow bandwidth of hollow cathode lamps makes spectral overlap rare. That is, it is unlikely that an absorption line from one element will overlap with another. Molecular emission is much broader, so it is more likely that some molecular absorption band will overlap with an atomic line. This can result in artificially high absorption and an improperly high calculation for the concentration in the solution. Three methods are typically used to correct this, namely Zeeman correction- A magnetic field is used to split the atomic line into two sidebands. These sidebands are close enough to the original wavelength to still overlap with molecular bands, but are far enough not to overlap with the atomic bands. The absorption in the presence and absence of a magnetic field can be compared, the difference being the atomic absorption of interest. Smith-Hieftje correction- The hollow cathode lamp is pulsed with high current, causing a larger atom population and self-absorption during the pulses. This self-absorption causes a broadening of the line and a reduction of the line intensity at the original wavelength [273]. Deuterium lamp correction- In this case, a separate source (a deuterium lamp) with broad emission is used to measure the background emission. The use of a separate lamp makes this method the least accurate, but its relative simplicity (and the fact that it is the oldest of the three) makes it the most commonly used method.

1.5 Statistical Treatment of Data

Analytical chemistry besides providing the methods and tools needed for insight into our material world [274], seeks to improve the reliability of existing techniques to meet the demands for better chemical measurements which arise constantly in our society [275]. Statistical analysis is necessary to understand the significance of collected data and to set limitations on each step of the analysis. The design of experiments is determined from proper understanding of what the data will represent. It is impossible to perform chemical analysis that is totally free from errors, or uncertainties. Every measurement is influenced by many uncertainties, which combine to produce a scatter of results. It is seldom easy to estimate the reliability of experimental data. However, the probable magnitude of the error in a measurement can often be evaluated statistically. Limits within which the true value of a measured quantity lies at a given level of probability can then be defined. Chemical analyses are affected by at least two types of errors namely systematic and random based on their source. Systematic errors have definitive value, assignable cause and unidirectional of nature. Systematic errors can be reduced to a negligible level if an analyst pays careful attention to the details of the analytical procedure including methods, periodic calibration of the instruments and self discipline. Random errors are the accumulated effect of the individual indeterminate uncertainties. It is caused by the uncontrollable variables that are an inevitable part of physical and chemical measurement. It causes the data to scatter more or less symmetrically around the mean in a random manner which are assumed to be distributed according to the normal error law (Gaussian curve). They are revealed by small differences in replicate measurements of a single quantity and affect the precision of the results. They are more difficult for an analyst to eliminate, but they can be minimized by increasing the number of replicate measurements. The random error in the result of an analysis can be evaluated by the method of statistics [276]. The most common applications of statistics to analytical chemistry include:  To establish confidence limits for the mean of a set of replicate data.  To determine the number of replicate measurements required to decrease the confidence limit for a mean to a given probability level.  To determine at a given probability whether an experimental mean is different from the accepted value for the quantity being measured (t test or test for bias in an analytical method). 35

 To determine at a given probability level whether two experimental means (different methods) are different.  To determine at a given probability level whether the precision of two sets of measurements differs (F test).  To decide whether a questionable data is probably the result of a gross error and should be discarded in calculating a mean (Q test).  To define an estimate detection limit of a method.

The commonly used terms in the statistical analysis Mean- The mean (x ) is obtained by dividing the sum of the replicate measurements

(Σxi) by the number of observations (N) performed in a set. The mean is considered to be the best estimate of the true value, which can only be obtained if an infinite number of measurements are performed. ∑ x x = i N Accuracy- The term accuracy as used in analytical chemical literature is a measure of the degree to which a mean (x ) obtained from a series of experimental measurements, agrees with the value, which is accepted as the true or correct value for the quantity. It is expressed by the error; either absolute or relative error. There are two types of errors according to their nature and source- systematic errors and random errors. Systematic error causes the mean of a set of data to differ (unidirectional) from the accepted value. They have definitive value, assignable cause and affect the accuracy of results. Random error causes data to be scattered more or less symmetrically around a mean value. It mainly affects the precision of measurement. Precision- Precision describes the agreement among the replicate measurements and is generally expressed as standard deviation, coefficient of variation (relative standard deviation) and variance. Standard deviation- Standard deviation(s) measures how closely the data are clustered about the mean. When measurements are repeated, the scatter of the results will be around the expected value of the results, if no bias exists.

2 Σ(x −x) s = i N −1 In analytical chemistry, this scatter is more often than not of such a nature that it can be described as a normal distribution. The mean gives the centre of the distribution. Standard deviation measures the width of the distribution. Therefore, an experimental technique

36 that produces a small standard deviation is more reliable (precise) than one that produces a large standard deviation provided that they are equally accurate. The square of the standard deviation, s2, is known as variance. Coefficient of variation- Precision is more often expressed as the coefficient of variation which is the standard deviation divided by the average and multiplied by 100. Since the average and standard deviation have same dimension, the coefficient of variation is dimensionless; it is only a relative measure of precision. Therefore, it is sometimes also known as relative standard deviation (RSD). s RSD = ×100 x Confidence limit and confidence interval- Calculation of s (standard deviation) for a set of data provides an indication of the precision inherent in a particular method of analysis. But unless there is a large number of data, it does not by itself give any information about how close the experimentally determined mean (x ) might be to the true mean value µ (population mean). Statistical theory, though, allows us to estimate the limits around an experimentally determined mean(x ) within which the population mean or true value (µ ) expected to lie, within a given degree of probability. The likelihood that the true value lies within the range is called confidence level or probability usually expressed as a recent. Confidence limit for µ = ± ts x N where t is the student’s t value which depends on the desired confidence level and this equation holds when σ, population standard deviation is not known. Correlation coefficient- The correlation coefficient is a measure of the linear relationship between two variables. In order to establish the linear relationship between variables xi and yi the Pearson’s correlation coefficient r is used. The r may have values between +1 nΣx y − Σx y r = i i i i Σ 2 − Σ 2 Σ 2 − Σ 2 [n xi ( xi ) ][n yi ( yi ) ] and –1. When the r is +1, there is a perfect correlation, i.e. an increase in one variable is associated with an increase in the other. When the r is –1, there is perfect negative correlation, i.e., one variable increases where as the other decreases. When r is zero, one variable has no linear relationship to the other. The correlation coefficient r is calculated by using the above equation.

Regression equation- When two variables exhibit a linear relationship, we may be interested in quantifying the relationship so that a value of one variable may be estimated from the other. A common example is the construction of calibration curve, which relates

nΣx y − Σx Σy b = i i i i 2 2 nΣx − (Σxi ) the concentration of analyte to absorbance or any measurable property. The least square curve fitting technique is the most commonly accepted mathematical procedure known for this purpose. The equation derived by this technique produces a line whose position is such that the sum of the squares of the vertical distances of each data point to the line is a minimum if the line is to be used to predict y from x values, or the sum of the squares of the horizontal distances is a minimum if x is to be predicted from y. If y = bx + a represents the equation for a straight line, where y is dependent variable and x is independent variable, then slope ‘b’ and intercept ‘a’ are derived from the following equations [276-278]. a = y − bx

Sensitivity- It is measure of the ability of an instrument or method to discriminate between small differences in analyte concentration. There are two factors which limit the sensitivity:  the slope of the calibration curve.  reproducibility or precision of the measuring device. Of two methods that have equal precision, the one that has the steeper calibration curve will be the more sensitive. A corollary to this statement is that if two methods have calibration curves with equal slopes, the one that exhibits the better precision will be the more sensitive. The quantitative definition of sensitivity that is accepted by the International Union of Pure and Applied Chemists (IUPAC) is calibration sensitivity, which is the slope of the calibration curve at the concentration of interest. Most calibration curves that are used in analytical chemistry are linear and may be represented by the equation: S = mc + Sbl, where S is the measured signal, c is the concentration of

the analyte, Sbl is the instrumental signal of the blank, and m is the slope of the straight

line. The quantity Sbl should be the intercept of the straight line. With such curves, the calibration sensitivity is independent of the concentration c and is equal to m. The calibration sensitivity as a figure of merit suffers from its failure to take into account the precision of individual measurements.

To include precision in a meaningful mathematical statement of sensitivity,

analytical sensitivity is proposed (γ): γ = m/ss; where ss = standard deviation of

measurement and m is the slope of the calibration curve [270,279].

Detection limits- It is defined as the minimum concentration or mass of analyte that can be detected at a known confidence level. This limit depends upon the ratio of the magnitude of the analytical signal to the size of the statistical fluctuations in the blank signal. The limit of detection (CL) according to the definition of International Union of

Pure and Applied Chemists can be expressed as [280]: CL = x + k.sbl ; where x = mean of

blank signal, sbl = standard deviation of the blank measures and k = numerical constant. A value of k=3 was strongly recommended by IUPAC. The Analytical Methods Committee of the Royal Society of Chemistry sought to clarify the IUPAC definition [281]. The estimation of the detection limit is best understood by considering a calibration graph. Using the linear regression method, it is possible to obtain the intercept and slope of the best-fit line. The value of the intercept can be used as x; and errors in the

slope and the intercept of the regression line is acceptable as a measure of sbl. Hence, the

detection limit (CL) may also be expressed as: CL = 3sbl/b; where b is the slope of the calibration line [270,277,282,283].

1.6 Present work and its scope

SPE has found its most effective use as a sample clean-up and concentration method prior to further analysis. Solid phase extraction, in combination with atomic absorption spectrometry, has been developed as the technique for preconcentration prior to determination of trace metal ions in varying complex matrices, which includes both biological and environmental samples. Amberlite XAD-4 and 16 resins have been successfully used as the polymeric supports in previous works (Table 1.2 and 1.3). Hence, we have used the same because of its good surface area (725 m2 g-1 and 800 m2 g-1) and high porosity. The functional groups used satisfactorily for this work are salicylic acid, o-hydroxybenzamide, salicylanilide and p-aminobenzene sulfonic acid on account of their smaller size and good number of chelating sites. This functionalities would render the modified resin more hydrophilic so as to facilitate faster equilibrium (of solute between solid and aqueous phases) whereby enhancing the extraction ability.

The modification of polystyrene-divinyl benzene adsorbent resin has been accomplished through azotization reaction, by conventional reaction techniques, by incorporating neutral organic hydrophilic group through –N=N- spacer. It is important to note that these functional groups are neutral, that is they bear no positive or negative charge but, however, they possess the characteristic features of a chelating agent. This is important in order to allow for effective contaminant removal of the inorganics since either anionic or cationic charged resins may often pick up undesirable materials that are present such as other matrices. However, neutral functionalized or modified resins such as those described here in act differently than charged resins and take up the inorganics by adsorbtion/chelation rather than ion exchange. Chelating resin and the aqueous solution containing the heavy metals along with other matrices may be contacted using both batch and column methods which would result in intimate contact between the resin and the solution. The columns used for extractions were packed with 100 to 500 mg of resin. Varying volume of aqueous solutions of a sample, containing metals in the concentration of the order of parts per million, is passed through the column at the optimum flow rate whereby the solute gets retained onto the resin. Investigations of the optimum experimental conditions sorption and elution have been carried out by considering the following influential parameters.

Synthesis and Characterization The introduction of the organic groups onto the polymeric material (XAD-series resin) can make the stable chelating compounds for the uptake of trace metal ions. The aim of chemical modification with different organic reagent, which has mainly N and O donor atoms, is to make the material have excellent coordination properties with trace metal ions and to obtain a novel sorbent with high loading of metal ions. The FT-IR analysis is a very useful technique in identifying the immobilization process by comparing the precursor and modified resin. The thermogravimetric analysis (TGA) curve of the chelating resin shows mass loss in two steps. In first step, it may be due to the loss of sorbed water of the resin and second step, due to the loss of functional moieties of the chelating resin. It was also use to performed, to study the water-regaining capacity of the resin matrix in order to evaluate its hydrophilic character. The CHN analysis was performed during each stage of chemical modification to study the extent of ligand functionalization to the polymeric matrix.

-1 The modified resin soaked in acidic (HCl/HNO3, 1-5 mol L ) and basic medium (NaOH, 1-4 mol L-1) for 6 h, filtered and then washed with triply distilled water until free from acid or alkali. The resin shows no loss in sorption capacity. Hence, the resin exhibited high chemical stability. The water regain capacity and hydrogen ion capacity have also been studied. This value reflects the high hydrophilicity of the resin which is satisfactory for column operation. In case of AXAD-16-SALD, the overall hydrogen ion capacity amounts to 6.08 mmol g-1 of resin, which may be contributed both by the hydroxylic and amide groups present within the molecule. Theoretically, if 2.65 mmol of the reagent constituted per gram of the resin, the hydrogen ion capacity, due to the hydroxylic group should have been 2.65 mmol g-1. The durability and reusability nature of the chelating resin was tested with metal ions solutions by batch equilibration method. Thereafter, the sorption and desorption of metal ions were repeated on the same resin. The capacity of the resin found to be constant up to the several cycles (35-55) showing the multiple use of chelating resin without any loss in its physical and chemical properties.

Sorption studies Effect of pH of the sample Sample pH is of prime importance for efficient retention of the trace elements on the sorbent. Its influence strongly depends on the nature of the sorbent used. Careful optimization of this parameter is thus crucial to ensure quantitative retention of the trace elements and in some cases selective retention. In particular with ion exchangers, correct adjustment of sample pH is required to ensure preconcentration. Thus, in the case of cationic-exchangers, low pH usually results in poor extraction due to competition between protons and cationic species for retention on the sorbent. When retention of trace elements is based on chelation (either in the sample or on the solid sorbent), the sample pH is also a very important factor as most chelating ligands are conjugated bases of weak acid groups and accordingly, they have a very strong affinity for hydrogen ions. The pH will determine the values of the conditional stability constants of the metal complexes. By contrast, pH may have no influence with some non-ionizable organic ligands [284]. For inorganic oxides, pH is also of prime importance. In particular, on amphoteric oxides such as TiO2 or Al2O3, cations are adsorbed at elevated pH due to the deprotonation of functional groups, whereas anion retention requires acidic conditions for the protonation of functional groups.

Adsorption isotherm The isotherm parameters of Langmuir and Frendlich for the sorption of metal ions have been studied. The results showed that the regression coefficients R2 obtained from Langmuir model were very close to 1, suggesting the Langmuir model could well interpret the studied adsorption procedure. From the comparison of correlation coefficients, it can be concluded that the data were fitted better by Langmuir equation than by Freundlich equation. Langmuir equation is applicable to homogeneous adsorption, where the adsorption of each adsorbate molecule onto the surface had equal adsorption activation energy. The fact shows that the adsorption of the hybrid sorbent is attributed to monolayer adsorption [285].

Kinetics of sorption In order to determine the uptake rate of metal ions on the synthesized resin and get access to the equilibrium time, studies on sorption kinetics were carried out. The sorbents characterized by good kinetic properties determined by the macroporous structure of the support, large surface area and total accessibility of the functional groups (without steric hindrance). The kinetic studies also showed that the temperature affected the rate constants significantly, that is, saturation was reached at a faster rate at higher temperature. This temperature effect may be a manifestation of the fact that the resin swells more completely at higher temperature, which allows metal ions to diffuse more easily into the interior of the resin, and that the sorption was an endothermic process and hence high temperature facilitates higher sorption. Moreover, both pseudo-first-order equation and pseudo second- order equation were used to express the sorption process of the chelating resin. The results showed that regression coefficients values (R2) of the pseudo-second-order model (>0.99) were better than those of the pseudo-first-order model for the sorbent, suggesting the pseudo-second-order model was more suitable to describe the sorption kinetics of chelating resin [285,286].

Sample flow-rate The sample flow-rate has been optimized to ensure quantitative retention along with minimization of the time required for sample processing. This parameter may have a direct effect on the breakthrough volume, and elevated flow-rates may reduce the breakthrough volume [284]. As a rule, cartridges and columns require lower maximum flow-rates than disks ranging typically from 0.5 to 5 mL min-1. This value may be

increased by a factor of 10 using disks. High flow rate has been found for the sorption of metal ions, such high flow rates support the superiority of present chelating resins.

Elution studies Nature of the eluent The nature of the eluent is of prime importance and should optimally meet three criteria: efficiency, selectivity and compatibility, as discussed below. In addition, it may be desirable to recover the analytes in a small volume of eluent to ensure a significant enrichment factor. The eluent may be an organic solvent (when reversed-phase sorbents are used), an acid (usually with ion-exchangers), or a complexing agent. Firstly, the eluting agent has been carefully chosen to ensure efficient recovery of the retained target species and quantitative recovery as far as possible [287]. A further characteristic of the eluent arises with the possibility of introducing selectivity. Using an eluent with a low or moderate eluting power, the less retained analytes can be recovered without eluting the strongly retained compounds. Thus, if the elements of interest are those that remain on the sorbent another elution step with a more eluent will ensure their quantitative recovery. In that way interfering analytes were removed during the first eluting step (also called washing step). On the opposite, if the compounds of interest are the less retained on the sorbent their elution with a low or moderate eluting agent ensures their selective recovery, as the interferent compounds will remain on the sorbent due to stronger interactions with the solid support. In some cases, this selectivity may favour speciation. For example, 1 mol L-1 HCOOH removed only Se4+ from an anion-exchange resin, leaving Se6+ retained on the sorbent, which was further eluted using 2 mol L-1 HCl [104]. Finally, the eluent should be compatible with the analysis technique. In particular, when using both flame

and electrothermal AAS, HNO3 has been preferred to other acids (namely H2SO4, HCl), as nitrate ion is a more acceptable matrix [288].

Effect of pH of eluent As retention of trace elements on solid sorbents is usually pH-dependent, careful choice of the eluent pH may enhance selectivity in the SPE procedure. As an example, once retained on Eriochrome black-T (ERT)-functionalized- silica gel, Mg2+ could be eluted first at a pH approximately 4, while increasing the pH to 5–6 was required for eluting Zn2+ [237].

Elution mode Most of the time, for practical reasons, sample loading and elution steps are performed in a similar manner. However, to avoid irreversible adsorption and ensure quantitative recoveries, elution in the back flush mode is recommended in some cases. This means that the eluent is pumped through the sorbent in the opposite direction to that of the sample during the preconcentration step. This is especially crucial when carbon- based sorbents have to be used due to possible irreversible sorption of the analytes.

Flow-rate of eluent The flow-rate of the elution was found to be high enough to avoid excessive duration, and low enough to ensure quantitative recovery of the target species. Typical flow-rates are in the range of 0.5 to 5 ml min-1 for cartridges/ columns and of 1 to 20 ml min-1 for disks [289]. As a rule, the higher the flow-rate, the larger the eluent volume required for complete elution [290,291].

Volume of Eluent The elution volume may be determined either experimentally or estimated theoretically [292]. The elution volume can usually be reduced by increasing the concentration of the eluent (e.g. acid). However, in this case, problems with subsequent analysis may be encountered (e.g. FAAS). Alternatively, the use of micro-sized disks may allow reduced solvent volume [293]. The elution step should enable sufficient time and elution volume to permit the metallic species to diffuse out of the solid sorbent pores. As a rule, 2 elution cycles are usually recommended as compared to a single step (e.g. two 5 ml elution should be preferred to a single 10 ml elution). Soaking time is also critical and 2 to 5 minute soak is most of the time allowed before each elution.

Breakthrough profile Sample volume to be percolated An important parameter to control in SPE is the breakthrough volume, which is the maximum sample volume that has been percolated through a given mass of sorbent after which analytes start to elute from the sorbent resulting in non-quantitative recoveries (Figure 1.5). The breakthrough volume is strongly correlated to the chromatographic retention of the analyte on the same sorbent and depends on the nature of both the sorbent and the trace element, as well as on the mass of sorbent considered and the analyte concentration in the sample [294]. In addition, it depends on the sorbent containers, as 44

disks usually offer higher breakthrough volumes than cartridges. This volume may be determined experimentally or estimated using several methods [292]. For that purpose the nature of the sample has to be taken into account, as the possible presence of ligands may dramatically reduce the breakthrough volume [295].

Figure 1.5 Typical representation of the breakthrough curve (i.e. concentration of the analyte at the outlet of the SPE system vs. sample volume percolated through the system).

VB is the breakthrough volume, VR the chromatographic elution volume, VC the sample

volume corresponding to the retention of the maximum amount of analyte and C0 is the initial analyte concentration in the sample. Preconcentration is a process in which the ratio of the quantity of a desired trace element to that of original matrix is increased but it does not necessarily mean increase in the concentration of the analyte. In order to demonstrate the preconcentration factor, preconcentration limit column procedure has been applied. The closeness of the dynamic capacity to the total sorption capacity reflects the applicability of the column technique for preconcentration.

Interference of sample matrix The presence of ligands in the sample matrix may affect trace element retention when stable complexes are formed in the sample with these ligands, as trace elements are less available for further retention. Thus, if metals are present in the sample as strong complexes, they may not dissociate resulting in no retention of the free metal on the sorbent. As an example, reduction in the retention of Cu2+ on Amberlite CG50 occurs in the presence of ligands such as glycine [296]. In the case of real samples, the presence of natural organic matter is of great concern as it may form complex with trace elements as observed for Cu2+ [296,297]. The most important class of complexing agents that occurs

naturally are the humic substances [298]. Their binding of metals through chelation is one of the most important environmental qualities. Yet, in some cases the presence of ligands may be a valuable tool for adding selectivity to the SPE step. This requires that the added ligands be correctly chosen to complex only the elements that are not of interest so that they are not retained on the sorbent [235]. The presence of ions other than the target ones in the sample may also cause problems during the SPE step. In particular, due to their usually high levels (e.g. Ca2+), they may hinder the preconcentration step by overloading the sorbent or cause interferences during spectrophotometric analysis. Therefore, their influence has been studied before validating a SPE method. Sometimes the addition of a proper masking agent (such as EDTA, thiourea or ethanolamine for example) may prevent the formation of interferences due to ions present in the sample [287]. Finally, the ionic strength of the sample is another parameter to control for an efficient SPE, as it may influence the retention of trace elements, and thus the value of the breakthrough volume for a given sorbent [299,300]. The chelating resin was found to tolerate high contents of various naturally occurring alkali and alkaline earth metals, anions and complexing agents in the determination of these metal ions.

Analytical method validation Great care has been taken to ensure that accurate results are obtained in the analysis. Every measurement has some imprecision associated with it, which results in random distribution of results. The experiment can be designed to narrow the range of this (confidence limit is set around the mean at some degree of probability), but it cannot be eliminated. Precision of the method is expressed either standard deviation or relative standard deviation. Systematic errors of instrumental and personal type are minimized by periodic calibration of the instruments and volumetric glassware, and self-discipline. However, systematic method errors are difficult to detect and introduces bias in the method. Bias of the analytical method has been estimated by the following procedures: Analyzing Standard Reference Materials (SRM) of biological, environmental and alloy type having complex sample matrix obtained from National Institute of Environmental studies (NIST), Iron and Steel institute of Japan (JSS) and National bureau of Standards (NBS). In order to demonstrate whether the difference of the observed mean of the replicate analysis of SRM and its certified value is due to merely the random error of the measurement or bias in the method, statistical t test is applied. Using a Second Independent and Reliable Analytical Method in parallel with the method being evaluated when standard samples are not available. The independent method should 46 differ as much as possible from the one under study. This minimizes the possibility that some common factor in the sample has the same effect on both methods. Then a statistical t and f test must be used to determine whether any difference is a result of random errors in the two methods or due to bias in the method under study. Performing Recovery Experiment which requires the spiking of the sample with known amounts of the analyte. The amount determined (recovered) by the method is expressed in terms of percentage recovery which shows accuracy of the developed method. By varying the sample size, the presence of a constant error can be detected.

Applications In order to explore the potential applicability of the chelating resin the developed methods were successfully applied for the determination of metal ions in natural and sewage water, mint leaves, mango pulp, fish, urine, multivitamins tablets, infant milk substitute and hydrogenated oil (ghee).

1.7 Merits of the present work

 The main advantage of the present procedure is the simple and fast preparation of the chelating resin and no requirement of organic solvents in the metal elution step.  The excellent ability for the exclusion of alkali and alkaline earth elements makes it desirable for use in the separation and preconcentration of trace elements because their presence often interfere in the subsequent FAAS determination.  The use of a column preconcentration technique allows for the assessment of low trace metal concentrations, even by less sensitive determination methods such as FAAS.  Preconcentration by this material from river water samples do not require any prior digestion of the samples. It can be successfully applied for the analysis of both environmental and biological samples as indicated by the high precision (low relative standard deviation). The designing and characterization of these four Amberlite XAD-4/16 based chelating resins and the investigation of their metal sorption behavior with their subsequent applications for analysis of various real samples containing varying complex matrices define the scope of the present thesis.

Table 1.2 Chelating agents used for modification of Amberlite XAD-4 resin Mode of Reagent Techniques coupled Metals Ref. preparation Chelate N-(dithiocarboxy)- sarcosine Spectrophotometry Co2+ 301 complex 2+ 8-Hydroxyquinoline (Oxine) Impregnation Spectrophotometry UO2 302 Chemically o-Aminobenzoic acid FAAS Pb2+,Cd2+,Ni2+, Co2+,Zn2+ 303 modified Chemically 2,3-Dihydroxy-naphthalene ICP-AES Cu2+, Ni2+, Co2+, Cd2+ 304 modified Ammoniumpyrrolidine- Cd2+,Cu2+,Mn2+,Ni2+,Pb2+, Impregnation ICP-AES 305 dithiocarbamate Zn2+ 2-Acetylmercapto- Chelate FAAS Cd2+,Co2+,Cu2+,Ni2+, Zn2+ 306 phenyldiazoaminoazobenzene complex 2,6-dihydroxyphenyl- Chelate FAAS Cd2+, Co2+, Cu2+, Zn2+ 307 diazoaminoazobenzene complex Chelate Cu2+, Fe2+, Pb2+, Ni2+, Diethyldithiocarbamates FAAS 308 complex Cd2+, Bi2+ Chelate Cd2+, Co2+,Cu2+, Ni2+, 1-Hydrazinophthalazine AAS 309 complex Pb2+, Fe2+, Cr3+Zn2+ 1-(2-pyridylazo)-2-naphthol Impregnation FI-FAAS Cu2+ 234 Chelate Cd2+, Cu2+, Ni2+, Pb2+, 1-(2-pyridylazo)-2-naphthol AAS 310 complex Cr3+, Mn2+ Chemically Salen AAS Cu2+, Pb2+, Bi2+ 311 modified Chemically Succinic acid Spectrophotometry U6+ 312 modified Chemically Cd2+, Co2+, Cu2+, Ni2+, Schiff bases FI–FAAS 313 modified Pb2+ Chelate O,O-Diethyldi-thiophosphate FI-FAAS Cd2+ 314 complex Chemically Rh3+ m-Phenylendi-amine ICP-AES 315 modified Chemically 2,3-Diamino-naphtalene Se6+ 316 modified Chemically 2-Aminothiophenol FAAS Cd2+, Ni2+ 317 modified

Table 1.3 Chelating agents used for modification of Amberlite XAD-16 resin Mode of Reagent Techniques coupled Metals Ref. preparation

1,6-bis(2-carboxy Chemically FAAS Cu2+, Cd2+ 318 aldehydephenoxy)butane modified 2,6-dichlorophenyl-3,3- 319 Impregnation FAAS Cu2+,Zn2+, Mn2+ bis(indolyl)methane Gallic acid Chemically Cr3+,Mn2+,Fe3+,Co2+, FAAS 320 modified Ni2+, Cu2+ 4-{[(2-hydroxyphenyl)imino] Chemically Zn2+, Mn2+, Ni2+, Pb2+, FAAS 164 methyl}-1,2-benzenediol modified Cd2+, Cu2+, Fe3+, Co2+

Acetylacetone Chemically AAS Cr 3+, Cr 6+ 321 modified 3,4-dihydroxy benzoyl methyl Chemically FAAS U6+ ,Th4+ 322 phosphonic acid modified Phthalic acid Chemically AAS Pb2+ 323 modified N,N-dibutyl-N_- Impregnation Spectrophotometry U6+ 160 benzoylthiourea (bis-2,3,4-trihydroxybenzyl) Chemically Spectrophotometry U6+, Th4+, Pb2+, Cd2+ 162 ethylene diamine modified Thiocyanate Impregnation FAAS Ag 2+ 324

1,5-diphenylcarbazone Impregnation FAAS Cr6+ ,Cr3+ 325

Nitrosonaphthol Chemically AAS Ni2+, Cu2+ 165 modified

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Chapter 2

Experimental

2.1 Instrumentation

Atomic absorption spectrometer GBC 932+ (Dandenong, Australia) atomic absorption spectrophotometer, equipped with hollow cathode lamp (and an air-acetylene flame) has been used for the determination of metal ions. Operating parameters set for the determination of elements are given in Table 2.1.

Table 2.1 Operating parameters set for FAAS for the determination of elements Flame composition Slit Wavelength Lamp Current Working range Air Acetylene Element Width (nm) (mA) (µg/ mL ) (L min-1) (L min-1) (nm) Co(II) 240.7 0.2 6.0 2.0-9.0 8.5 2.3 Ni(II) 232.0 0.2 4.0 1.8-8.0 8.5 2.3 Cu(II) 324.8 0.7 3.0 2.5-10.0 8.5 2.3 Zn(II) 213.9 0.7 5.0 0.4-1.5 8.5 2.3 Cr(III) 357.9 0.7 6.0 2.0-15.0 8.5 2.3 Mn(II) 280.1 0.2 5.0 1.4-5.5 8.5 2.3 Cd(II) 228.8 0.7 3.0 0.2-1.8 8.5 2.3 Pb(II) 283.3 0.7 5.0 7.0-50.0 8.5 2.3 Fe(III) 248.3 0.2 7.0 2.0-9.0 8.5 2.3

Fourier Transform-IR Spectrometer All the infrared (IR) spectra were recorded on a Fourier Transform-IR Spectrometer from Spectro Lab-Interspec 2020 (Newbury, UK) using KBr disc method. Significance in context to our work: The IR spectra of the modified resins (both metal loaded as well as metal free resins) have been taken. The IR spectra of each of the modified chelating resins exhibited bands that support the immobilization of the ligands onto the polymeric support, Amberlite XAD-4 and XAD-16. The IR spectrum of each intermediate favors the proposed synthetic route (Scheme 2.1) for the chelating resins. The diazotization and the subsequent immobilization of the ligands, namely Salicylic acid (SA), o-Hydroxybenzamide (HBAM) onto Amberlite XAD-4 75

and Salicylanilide (SALD), p-Aminobenzene sulfonic acid (ABSA) onto Amberlite XAD-16

occur most probably at para position with respect to –CH-CH2-. The probability of occurring at ortho position of the polystyrene ring is not very probable owing to electronic effects and steric hindrance.

TG/DTA simultaneous measuring instrument A Shimadzu TG/DTA simultaneous measuring instrument, DTG-60/60H (Kyoto, Japan) was used for the thermogravimetric analysis (TGA) and differential thermal analysis (DTA). Significance in context to our work: The thermal stability was checked by performing thermogravimetric analysis of Amberlite XAD-4 modified with SA, HBAM and Amberlite XAD-16 modified with SALD and ABSA. The TGA curves of all the chelating resins show an initial weight loss up to 140- 220 °C which may correspond to the loss of weakly bonded water molecules. The capability of these resins to hold water molecule makes them suitable for enrichment of metal ion from extremely dilute solutions.

Elemental analyzer (CHN) In the present work, CHN analysis was carried out with Carlo Erba EA1108 (Milan, Italy) elemental analyzer (available at SAIF, CDRI, Lucknow, India). Significance in context to our work: The extent of the coupling reaction may be interpreted from the composition of the final resin. The total carbon, nitrogen and hydrogen contents can give a good approximation of the quantity of reagent incorporated through azo spacer. Hence, the synthesized resins were subjected to elemental (CHN) analysis. CHN analysis of the products formed during the intermediate steps was also carried out in order to monitor the course of the reactions. The results are compared with the theoretical values to get a better interpretation of the composition of the products. These results in association with TGA suggest that the chelating ligand immobilized onto repeat unit of polymer is very close to 1:1 ratio and approximately one to one and a half water molecule per repeat unit of polymer are present in all the three chemically bonded chelating resins.

Mechanical shaker In the present study, a thermostated mechanical shaker (Narang Scientific works, New Delhi, India), equipped with digital display (±0.2°C), with shaking speed variable from 10 to 200 strokes per minute and supplied with gabled cover and lotus clamps for conical flask (5 nos.) for 100-500mL flasks, was used for carrying out equilibrium studies.

Chromatographic column A short glass column with an inner diameter of 10 mm and a length of 100 mm, provided with porous frits was obtained from J-SIL Scientific industries, Agra, India. The column was filled with a suspension of specified amount of resin in water (Figure 2.1).

2.2 Reagents and solutions

Standard solutions of metal ions The stock solutions (1000 mg L-1) of Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Pb(II) were prepared by dissolving appropriate amount of their nitrate or chloride salts in water acidified with 10 mL of corresponding concentrated acid. All the metal salts were supplied by Central Drug House (P) Ltd., New Delhi and were of analytical reagent grade. The solutions were standardized by complexometric titration [1, 2] before use and diluted according to requirement. The analytical reagent grade of nitric acid (15.4 mol L-1) and hydrochloric acid (11.6 mol L-1) were diluted to lower concentrations of 0.001-5.0 mol L-1 with triply distilled water. The solution of NaOH was prepared by dissolving an appropriate amount in 1000 mL. It was standardized titrimetrically before use. All the reagents (HNO3, HCl, HClO4 and H2O2) used for wet digestion of the samples were procured from Merck (Mumbai, India).

Buffer solutions Buffers covering the pH range (1.0-2.2) were prepared by mixing appropriate amount of KCl (0.2 mol L-1) and HCl (0.2 mol L-1) [3]. Buffers for pH 3.72-5.57 were prepared by mixing acetic acid (0.2 mol L-1) and sodium acetate (0.2 mol L-1) solutions in the appropriate ratio [4]. Appropriate mixing ratio [5] of 0.1 mol L-1 Boric acid solution (0.1 mol L-1 KCl)

and NaOH solution (0.1 mol L-1) were used to prepare buffer for pH 8.0. For buffer of pH -1 -1 9.0-10, 0.2 mol L NH4Cl solution was mixed with 0.2 mol L NH3 [6].

Figure 2.1 Diagram depicting a preconcentration system

Dilution of nitric acid, hydrochloric acid and sodium hydroxide solution

Polymeric support Non-ionic polystyrene-divinylbenzene co-polymers, Amberlite XAD-4 (20-60 mesh particle size with 725 m2 g−1 of surface area) and Amberlite XAD-16 (20-60 mesh particle size with 800 m2 g−1 of surface area) resin (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were purchased. The resin was pretreated with an ethanol-hydrochloric acid-water (2:1:1) solution for overnight and subsequently rinsed with distilled water until pH of the supernatant water became neutral so that it becomes free from any impurities.

Chelating ligands Salicylic acid (SA), o-Hydroxybenzamide (HBAM), Salicylanilide (SALD) and p- Aminobenzene sulfonic acid (ABSA) were procured from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). They were used without any further purification.

Standard reference materials Standard reference materials (with composition as illustrated in Table 2.2) such as vehicle exhaust particulates NIES 8, Pond sediment NIES 2, Chlorella NIES 3, Human hair NIES 5, Tea leaves NIES 7 were obtained from the National Institute of Environmental Studies (Ibaraki, Japan), Rompin hematite JSS (800-3) obtained from the Iron and Steel Institute of Japan (Tokyo, Japan) and Zinc base die-casting alloy C NBS 627 provided by the National bureau of Standards, U.S. Department of Commerce, (Washington, DC, USA).

2.3 Pretreatment of samples

2.3.1 Collection of water samples The water samples namely river water (collected from the Ganga, Narora.), canal water (collected from Kasimpur, Aligarh), sewage water (collected from area in the vicinity of local nickel electroplating industry, Aligarh) and tap water (collected from University campus) were immediately filtered through millipore cellulose membrane filter (0.45 μm pore size), acidified to pH 2 with concentrated HNO3 (obtained from Merck, Mumbai, India), and stored in pre-cleaned polyethylene bottles. The bottles were cleaned by soaking in an alkaline detergent, 4 mol L–1 HCl and finally treated with 2 mol L–1 HF and 0.5 mol L–1

HNO3, and rinsed with triply distilled water between each step.

2.3.2 Digestion of multivitamin samples, infant milk substitute and vanaspati ghee A multivitamin capsule (bearing the commercial name Maxirich) was procured from Cipla Limited (Mumbai, India). For the experiment, five multivitamin capsules (5.64 g) were

taken in a beaker containing 25 mL of concentrated HNO3 and digested by slowly increasing the temperature of the mixture to 120 ºC. The mixture was further heated till a solid residue

was obtained. It was allowed to cool and then dissolved in 20 mL of concentrated HNO3. The solution was gently evaporated on a steam bath until a residue was left again. It was

subsequently mixed with 50 mL of distilled water and concentrated HNO3 was then added drop wise until a clear solution was obtained on gentle heating. Infant Milk substitute (commercially available as Lactogen 1) was obtained from Nestle India Limited (New Delhi, India). An amount of 200 mg was heated in a beaker

containing mixture of concentrated H2SO4 (20 mL) and HNO3 (10 mL) till a clear solution

was obtained. It was allowed to cool and most of the acid was neutralized with NaOH. The total volume was made up to 50 mL and kept as stock. Hydrogenated oil (locally known as Vanaspati ghee) was obtained from the local market, Aligarh, India. For the experiment, 2 g was taken in a beaker and dissolved in 15mL of concentrated nitric acid with heating. The solution was cooled, diluted and filtered. The filtrate was made up to 50 mL with deionized water after adjusting its pH to the optimum value.

Table 2.2 Composition of standard reference materials Environmental and biological Certified value (µg g-1) Samples Co: 3.3, Cu: 67, Ni: 18.5, Pb: 219.0, Zn: 1040, NIES 8 Cr:25.5, Cd:1.1, Ca: 5300, Mg:1010, K: 1150, Na:1920, P:510 Zn: 343.0, Ni: 40.0, Co: 27, Cu: 210.0, Cd: 8200, NIES 2 Cr:75, Ca: 8100, Fe: 65300, K:6800, Na: 5700, P:1400 National Institute of Zn: 20.5, Cu: 3.5, Co: 0.87, Pb: 0.60, Fe:1850, Environmental studies NIES 3 Ca:4900, Mn:69, Mg:3300, K:12400, P:17000 (NIES) Zn: 169.0, Cu: 16.3, Co:1000, Ni: 1.8, Pb: 6.0, NIES 5 Cr:14000, Ca:728, Fe: 225, Mg: 208, K:34, Na:26, Ba: 3.2, P:165 Zn:33.0, Cu: 7.0, Co:0.12, Cr:0.15, Ni:6.5, Cd:300, NIES 7 Ca:3200, Pb:8000, Mg: 1530, Mn: 700, K: 18600, Na: 15.5, Ba: 5.7, P:3700 Iron and Steel Institute of JSS Cu: 640.0, Zn: 1030.0, Pb: 210.0, Sn:120, Mn:2200, Japan (JSS) (800-3) Bi: 230, P:420, S:740 National bureau of NBS Cu: 1320.0, Pb:82.0, Ni: 29.0, Sn:42 Fe: 230, Cr:36, Standards (NBS) 627 Mg: 300, Si: 210, Cd: 51, Al:38800

2.3.3 Digestion of standard reference materials To dissolve the environmental SRMs (Vehicle exhaust particulates NIES 8 and Pond sediment NIES 2), a 0.5 g of the sample was dissolved by adding 10 mL of concentrated 80

nitric acid (15.5 mol L-1), 10 mL of concentrated perchloric acid (12.2 mol L-1) and 2 mL of concentrated hydrofluoric acid (22.4 mol L-1) in a 100 mL in a Teflon beaker. The solution was evaporated to near dryness, redissolved in minimum volume of 2% HCl, filtered and made up to 50 mL volume in a calibrated flask. The sample solutions of biological SRMs (Human hair NIES 5 and Tea leaves NIES 7) were prepared as proposed by the international atomic energy agency [7]. A 50 mg (600 mg for Chlorella NIES 3) of each of the samples was agitated with 25 mL of acetone, and then washed three times with distilled water and with 25 mL of acetone. The contact time of the cleaning medium with the sample was 10 min. The samples were finally dried for 16 h at 100 °C. Then each of the samples was dissolved in 10-20 mL of concentrated nitric acid.

After adding 0.5 mL of 30% H2O2, the solution was boiled to dryness. The residue obtained was dissolved in minimum amount of 2% HCl and made up to a 50 mL volume in a calibrated flask. The solution of standard alloys (Rompin hematite JSS (800-3) and Zinc base die- casting alloy C NBS 627) was prepared by taking 25 mg of the sample into a beaker and dissolved in 10-50 mL of aqua–regia. The solution was boiled to near dryness. Finally the residue was dissolved in minimum volume of 2% HCl and filtered through a Whatman filter paper No.1. The residue was washed with two 5 mL portions of hot 2% HCl. The aqueous layer was evaporated to dryness. The residue was redissolved in 5 mL of 2% HCl and made up to 50 mL with distilled water.

2.3.4 Digestion of fish and urine samples A total of 10 fishes (weighing 35.70 ± 0.60 g each), Common carbs (Cyprinus carpio) were caught from different locations of the river Ganga (Narora, Aligarh). Fish were dissected to separate organs (flesh, gills, liver and kidney) according to FAO methods [8]. The separated organs were put in Petri dishes to dry at 120 °C until a constant weight was reached. The separated organs were placed into digestion flasks and ultrapure concentrated nitric acid and hydrogen peroxide (1:1 v/v) was added. The digestion flasks were then heated to 130°C until all the materials were dissolved. The digest was diluted with double distilled water (50 mL) for further experiments.

A 10 mL portion of urine sample was treated with 10 mL of concentrated HNO3 and

a HClO4 mixture of 2:1 in a 100 mL beaker covered with a watch glass. The contents in the

beaker were heated on a hot plate (100°C 15 min, 150°C 10 min). The watch glass was removed and the acid evaporated to dryness at 150°C. To the obtained white residue, 10 mL

of HClO4 was added, and the mixture was heated at 160°C to dryness. All of the heating processes were carried out under a hood with necessary precautions. Six milliliters of 1 M HCl were added, and the contents were heated at 150°C for 2 min. The pH of the obtained clear solution was adjusted to 4 by a 3 M NaOH solution. The digest was diluted with distilled water (50 mL) for further experiments.

2.3.5 Digestion of mango pulp and mint leaves Mango pulp was purchased from the local market of Aligarh. An amount of 25 g of mango pulp was taken in to a 100 mL volumetric flask, containing 15 mL of HCl. The mixture was shaked and subsequently subjected to centrifugation, followed by filtration in order to remove the suspended solid matter. The pH of the solution was adjusted to 4.0 with appropriate buffer solution and the volume was made up to 100 mL before applying it to the recommended column procedure. Leaves of mint –a perennial herb (Mentha arvensis), procured from Aligarh market, were repeatedly washed with triply distilled water before drying in an oven (at 70 °C) and subsequently ground to fine powder following which it was sieved through 1 mm nylon mesh. A portion of 1 g of the sample was digested with 5 mL of a mixture nitric acid and perchloric acid (in the ratio of 3:2) at 110 °C for 5h. The digested samples were then diluted with triply distilled water and made up the volume of 50 mL before filtering it through Whatmann filter paper and subsequently subjecting to the recommended column procedure.

2.4 Preparation of chelating resin

Chelating resins based on organic polymer may have functional group in their polymeric skeleton or these may be modified by chelating ligands, which are immobilized by physical adsorption or chemical bonding. The anchoring of ligands on these polymers through covalent bonding is attained, either by directly linking the chelating ligand covalently or via a spacer. The methylene spacer [9, 10] and azo spacer [11, 12] are commonly used. The immobilization of ligands through covalent linkage on the polymer matrix as a pendant group with or without spacer offers much wider ranges of possibilities in

fabricating chelating resins and consequently their tailoring for application such as a preconcentrating matrix for metal ions becomes possible. The coupling of Salicylic acid (SA), o-Hydroxybenzamide (HBAM), Salicylanilide (SALD) and p-Aminobenzene sulfonic acid (ABSA) with Amberlite XAD-4 and Amberlite XAD-16 were attempted by this method using azo spacer. The preparation and characterization of these resins are detailed below.

Scheme for synthesis of Amberlite XAD-4 and XAD-16 based resins The Amberlite XAD-4 and Amberlite XAD-16 after cleansing were subjected to nitration and subsequently the nitro group was reduced to get the amino polymer. It was diazotized and the resulting diazonium salt was coupled with four chelating ligands namely Salicylic acid (SA), o-Hydroxybenzamide (HBAM), Salicylanilide (SALD) and p- Aminobenzene sulfonic acid (ABSA) separately to synthesize the corresponding chelating resins. The experimental details are given below and various synthetic steps are schematically shown in Scheme 2.1.

Nitration of Amberlite XAD-4 and XAD-16 Amberlite XAD-4 and Amberlite XAD-16 beads (5 g) were mixed with a nitrating mixture (25 mL conc. H2SO4 and 10 mL of conc. HNO3) in a 250 ml round bottomed flask placed in a thermostatically controlled heating mantle at 50 °C. The mixture was stirred for 20 min at this temperature. Thereafter, the nitrated resin was poured into an ice-cold water bath and filtered. It was repeatedly washed with distilled water until it became free from acid.

Synthesis of amino resin

The reduction of the nitrated resin was carried out by refluxing it with SnCl2 (40 g) concentrated HCl (45 mL) and ethanol (50 mL) for 12 h with constant stirring. The amino- resin-tin complex was filtered and washed with distilled water and subsequently it was treated with excess of 2 mol L-1 NaOH solution to release free amino resin. According to the following reaction:

(CH CH2)n (CH CH2)n concentrated HNO3 + H2SO4

NO2 (i) SnCl2/HCl in EtOH (ii) NaOH (CH CH2)n

NH2 NaNO2 + HCl 0-5 °C (CH CH2)n

N2Cl 10% NaOH

N N N N N N N N NH OH OH 2 NH2 H N OH O SO3H OH O O

-CH-CH - 2 -CH-CH2- -CH-CH2- -CH-CH2-

N N N N N N N N NH2 OH H OH NH2 N SO3H OH O OH O O

XAD-4-SA XAD-4-HBAM XAD-16-ABSA XAD-16-SALD Scheme 2.1 Synthesis of chelating resins by incorporating reagents through azo spacer.

R (NH3)2SnCl6 + NaOH = 2 RNH2 + Na2SnO3 + 6 NaCl + 5 H2O

It was filtered and treated with 2 mol L-1 HCl to convert it into hydrochloride. Thereafter, it was washed with doubly distilled water to remove excess of HCl.

Diazotization of amino resin The hydrochloride form of the amino resin (5 g) was suspended in 200 ml of ice cold -1 -1 water and then diazotized with 1 mol L HCl and 1 mol L NaNO2 solution at 0-5 °C until the reaction mixture started to change the colour of iodide paper to violet. The diazotized polymer was filtered under ice cold condition in order to prevent the disintegration of the diazotized compound at higher temperature.

Synthesis of Amberlite XAD-4 and Amberlite XAD-16 immobilized with respective chelating ligands The diazotized resin under cold condition was reacted with the respective ligand (5 g taken in 250 ml of 10% NaOH solution) at 0 to 5 °C for 24 h and the resulting beads were filtered and washed with 4mol L-1 HCl and doubly distilled water successively and finally

dried at 50 °C and kept over fused CaCl2 in a desiccator for further use.

2.5 Characterization of chelating resins

2.5.1 Water regain value The rate of metal ion phase transfer is governed by the extent of hydrophilicity of the polymeric matrix. Water regain is defined as the amount of water absorbed by 1.0 g of polymer. The dried resin was stirred in doubly distilled water for 48 h, and then filtered off by suction, dried in air, weighed, dried again at 100 °C overnight and reweighed. The water

regain value was calculated as: W = (mw – md)/ md, where mw is the weight of the air-dried

polymer after filtration by suction and md is the weight of the resin after drying at 100 °C overnight.

2.5.2 Hydrogen ion capacity For overall hydrogen ion capacity, an accurately weighed (0.5 g) resin was first treated with 4.0 mol L-1 HC1 and then filtered off, washed with distilled water to make it free from acid and dried at 100 °C for 5–6 h. The acidic form of the resin was equilibrated with 20.0 mL of 0.1 mol L-1 NaOH solution for 6 h at room temperature at stirring condition and then the excess alkali was estimated with 0.1 mol L-1 hydrochloric acid solution. In order to 85

evaluate the contribution of the hydrogen ions constituting the stronger acid (functional group) to the overall hydrogen ion capacity, another sample of the resin in the acid form was

equilibrated with NaHCO3 solution in place of NaOH.

2.6 Recommended procedure for sorption studies of metal ions

2.6.1 Batch ‘static’ method A weighed amount of the synthesized/modified resin was equilibrated with suitable volume of metal solution of appropriate concentration maintained at constant pH for 2 h. The resin was filtered and the sorbed metal ions were desorbed by shaking with the appropriate

eluent and subsequently analyzed by FAAS.

Effect of pH To determine the optimum pH of metal ion uptake, excess of metal ion (50 mL, 100 µg mL-1) was shaken with 100 mg of resin for 120 minutes. The pH of metal ion solution was adjusted prior to equilibration over a range pH 2-10 with the corresponding buffer system. pH >10 was not studied to avoid metal hydroxide precipitation.

Kinetics of sorption For studying the effect of time on the sorption capacity, a 0.1 g amount of resin beads was stirred with 100 mL of solution containing one of the metal ions (20 µg mL-1) at two different temperatures for 2, 5, 10, 20, 30 ,40, 60, 80, 100 and 120 min (under the optimum conditions). The kinetic studies also showed that the temperature affected the rate constants significantly; that is, saturation was reached at a faster rate at higher temperature. This temperature effect may be a manifestation of the fact that the resin swells more completely at higher temperature, which allows metal ions to diffuse more easily into the interior of the resin, and that the sorption was an endothermic process and hence high temperature facilitates higher sorption. A plot of log D versus 1/T, where the distribution ratio (D) represents the ratio of sorption capacity and the concentration of free metal ion at the equilibrium sorption, respectively, revealed that the distribution ratio increased with the increase of temperature. This again implies that the sorption process was an endothermic

process. The values of ΔH and ΔS were calculated using the slope and intercept from the above plots using the following relationship [13,14]: − ∆H ∆S log D = − + (1) 2.303RT 2.303R The dynamics of the adsorption process in terms of the order and the rate constant can be evaluated using the kinetic adsorption data. The process of the mention metal ions removal from an aqueous phase by any adsorbent can be explained by using kinetic models and examining the rate-controlling mechanism of the adsorption process such as chemical reaction, diffusion control and mass transfer. The kinetic parameters are useful in predicting the adsorption rate which can be used as important information in designing and modeling of the adsorption operation. The kinetics of removal of such ions is explicitly explained in the literature using pseudo first-order, second-order kinetic models [14-16]. k t Pseudo-first-order model: log(q − q ) =log q − 1 (2) e t e 2.303 t 1 t = + Pseudo-second-order model: 2 (3) qt k2 q e qe

Where k1 and k2 is the rate constant of pseudo first-order and pseudo-second- order

rate constant, respectively. qe and qt are adsorption capacity at equilibrium at anytime, respectively.

Effect of resin amount To investigate the effect of the amount of the resin on the sorption of metal ions, an excess of the metal ion solution corresponding to 150 µg mL-1 was equilibrated with varying amounts of resin buffered at the optimum pH.

Langmuir and Freundlich isotherm study For an adsorption column, the column resin is composed of microbeads. Each binding particle immobilized to the micro bead can be assumed to bind in a 1:1 ratio with the solute sample passed through the column. The Langmuir model assumes that sorption occurs on defined sites of the sorbent with no interaction between the sorbed species and that each site can accommodate only one molecule (monolayer adsorption) with the same enthalpy

sorption, independent of surface coverage. The linearized form of Langmuir isotherm may be represented by the following equation [15-17].

Ce 1 Ce = + q q b q e m m (4) -1 Here, Ce is the equilibrium concentration (mg L ), qe is the amount adsorbed at equilibrium -1 (mg g ) and qm and b is Langmuir constants related to adsorption efficiency and energy of adsorption, respectively. The Freundlich isotherm postulates heterogeneous surface energy system and hence is the most convenient form of representing the experimental data obtained for different particle sizes [15, 18]. Therefore, the Freundlich isotherm was also modeled to fit the equilibrium data and is given by the following equation

ln qe = ln KF + ln Ce (5)

where KF and 1/n are Freundlich constants related to adsorption capacity and energy of adsorption, respectively.

2.6.2 Column ‘dynamic’ method A sample of synthesized/modified resin was soaked in water for 24 h and then poured into a glass column (1 × 10 cm). The resin bed in the column was further buffered with 5 mL of the appropriate buffer system. A solution of metal ions of optimum concentration was passed through the column at an optimum flow rates after adjusting to a suitable pH with suitable buffers. After the sorption operation, recovery experiments were performed; for this purpose the column was washed with water and then appropriate volume of the proper eluent was made to percolate through the bed of loaded resin whereby the sorbed metal ions get eluted. The eluents were collected in 5 mL for the subsequent determination by FAAS.

Effect of flow rate for sorption The effect of flow rate on the sorption was studied by varying the flow rate 2–8 mL min−1 at the pH chosen for maximum sorption, keeping a constant column height.

Effect of matrix Various cations and anions, which are inevitably associated with heavy metals, may interfere in the latter’s determination through precipitate formation, redox reactions, or 88

competing complexation reactions. Common chemical species such as sodium citrate, 2- 2- + 2- 2- sodium tartrate, sodium oxalate, humic acid, fulvic acid, NO3 , CO3 , NH4 , SO4 , PO4 , Cl–, K+ and Na+ were checked for any interference in the sorption of these metals. In order to determine the tolerance limit of the resin for various interfering electrolytes and metal ion species, studies were carried out using metal ion solutions (100 mL, 10 µg L-1) allowed to percolate individually through a column packed with 100 mg of resin with varying amounts of electrolyte or metal ions till interference was observed.

Preconcentration factor and breakthrough capacity The lower limit of quantitative preconcentration below which recovery becomes non- quantitative (preconcentration limit) was determined by increasing the volume of metal ion solution and keeping the total amount of loaded metal ion constant at 10 μg. The breakthrough volume, which corresponds to the volume at which the effluent concentration of any chemical species from the column is about 3–5% of the influent concentration, was determined by applying the recommended procedure to varying volume of the metal ion solution. The effluent fractions were collected in 5 mL and analyzed for the presence of the metal.

Calibration and detection limit The standard solutions for calibration, for each metal, were prepared in 100 mL by taking suitable aliquot of metal ions and buffer solutions, and then subjected to the recommended column procedure. A blank run was also performed applying recommended column procedure with the same volume of aqueous solution prepared by adding suitable buffer (excluding metal ions) and finally eluting the same in 5 mL before subjecting it to FAAS determination.

2.6.3 Procedure for desorption studies Type of eluting agents and effect of flow rate

Recovery studies were performed with different mineral acids namely H2SO4, HCl and HNO3. The efficiency of stripping was studied by using different volumes (1-10 mL) and concentrations (0.1-5.0 mol L-1) of the mineral acids.

The effect of flow rate on the sorption was studied by varying the flow rate 2–8 mL min−1 of each mineral acid and subsequently determining the amount recovered.

Resin reusability test The re-usability of the resin was tested by loading the metal ions several times on a column from a solution having a concentration 50 µg mL-1 at a flow rate of 2 mL min-1 and eluting by the appropriate eluting agent.

2.7 Procedures for method validation

Validation of the method was carried out by employing metal ion standard solutions and standard reference materials. Recoveries of selected trace metal ions were ascertained using single and mixed metal ion standard solutions.

Precision Using optimum conditions, the precision of the method was evaluated by carrying out six successive sorption and elution cycles of 10 µg each of the metal studied taken in 100 mL following the recommended procedure. The relative standard deviations (RSD) for the observed values were then evaluated. The present method was applied for the analysis of various SRMs including environmental, biological and alloy samples. The percentage recovery was subsequently evaluated for precision.

Accuracy The accuracy of the present method was evaluated from the results of the analysis of various SRMs including environmental, biological and alloy samples. Student’s t-test was performed to compare the observed mean concentration values of the metals studied with the certified values. The validity of the results was tested by standard addition method, by spiking a known amount of individual metal ions to the water samples. The recovery of the analytes was determined subsequently.

References

[1] A.I. Vogel, A textbook of quantitative inorganic analysis, Longman London, Fourth edition, 1978, p. 319.

[2] F.J. Welcher, The analytical uses of ethylenediaminetetraacetic acid, Van Nostrand company Inc., Princeton, New jersey, New York, 1958.

[3] G. Gomori, Preparation of buffers for use in enzyme studies, Methods Enzymol. 1 (1955) 138.

[4] H.T.S. Britton, Hydrogen ions: Their determination and importance in pure and industrial chemistry, E.H. Tripp (Ed), Vol. 1, Chapman and Hall Ltd, London, 1956.

[5] W.M. Clark, H.A. Lubs, Hydrogen electrode potentials of phthalate, phosphate and borate buffer mixture, The Journal of Biological Chemistry 25 (1916) 479.

[6] J.A. Dean (Ed), Lange’s Handbook of chemistry, Fifteenth edition, McGraw Hill, New York, 1999.

[7] J. Kubova, V. Hanakova, J. Medved, V. Stresko, Determination of lead and cadmium in human hair by atomic absorption spectrometric procedures after solid phase extraction, Anal. Chim. Acta 337 (1997) 329.

[8] B. Dybem, Field sampling and preparation subsamples of aquatic organism for analysis metals and organochlorides, FAO. Fisher Tech. 212 (1983) 1.

[9] V.A. Lemos, P.X. Baliza, J.S. Santos, L.S. Nunes, A.A. de Jesus, M.E. Rocha, A new functionalized resin and its application in preconcentration system with multivariate optimization for nickel determination in food samples, Talanta 66 (2005) 174.

[10] R.S. Praveen, P. Metilda, S. Daniel, T. Prasada Rao, Solid phase extractive preconcentration of uranium(VI) using quinoline-8-ol anchored chloromethylated polymeric resin beads, Talanta 67 (2005) 960.

[11] A. Islam, M.A. Laskar, A. Ahmad, Characterization of a novel chelating resin of enhanced hydrophilicity and its analytical utility for preconcentration of trace metal ions, Talanta 81 (2010) 1772.

[12] A. Islam, M.A. Laskar, A. Ahmad, The efficiency of Amberlite XAD-4 resin loaded with 1-(2-pyridylazo)-2-naphthol in preconcentration and separation of some toxic metal ions by flame atomic absorption spectrometry, Environ. Monit. Assess. 175 (2011) 201-212.

[13] A. Islam, M.A. Laskar, A. Ahmad, Characterization and Application of 1-(2- Pyridylazo)-2-naphthol Functionalized Amberlite XAD-4 for Preconcentration of Trace Metal Ions in Real Matrices, J. Chem. Eng. Data 55 (2010) 5553. 91

[14] K.G. Sreejalekshmia, K. Anoop Krishnanb, T.S. Anirudhana, Adsorption of Pb(II) and Pb(II)-citric acid on sawdust activated carbon: Kinetic and equilibrium isotherm studies, J. Hazard. Mater. 161 (2009) 1506.

[15] M.H. Kalavathy, L.R. Miranda, Moringa oleifera-A solid phase extractant for the removal of copper, nickel and zinc from aqueous solutions, Chem. Eng. J. 158 (2010) 188.

[16] Y. Tiana, P. Yina, R. Qua, C. Wanga, H. Zhengb, Z. Yua, Removal of transition metal ions from aqueous solutions by adsorption using a novel hybrid material silica gel chemically modified by triethylenetetraminomethylenephosphonic acid, Chem. Eng. J. 162 (2010) 573.

[17] I. Langmuir, The adsorption of gases on plane surfaces of glass, mica and platinum, J. Am. Chem. Soc. 40 (1918) 1361.

[18] C. Namasivayam, D. Kavitha, Removal of Congo Red from water by adsorption onto activated carbon prepared from coir pith, an agricultural solid waste, Dyes Pigm. 54 (2002) 47.

Chapter 3

Characterization of a chelating resin functionalized via azo spacer and its analytical applicability for the determination of trace metal ions in real matrices

3.1 Introduction

The use of chelating resin in solid-phase extraction (SPE) as metal ion extractant has attracted researchers in the field of separation science [1-3]. The possibility to extract selectively a number of analytes over a wide pH range, quantitative sorption and elution, kinetically faster sorption and desorption mechanisms, good retention capacity, high preconcentration factor and regeneration of resins over many cycles with good reproducibility in the sorption characteristics are frequently quoted as an advantage [3]. Activated carbon [4], polymeric fibers [5], Ambersorb [6], inorganic ion-exchanger [7], alumina [8] and silica gel [9] have been used to preconcentrate trace metal ions. However, they suffer from lack of selectivity, which leads to high interference of other existing species with the analyte metal ion and chemical stability [10]. Amberlite XAD series resins with polystyrene-divinyl benzene copolymer matrix have proved themselves as efficient support for anchoring chelating ligands due to uniform pore size distribution, high surface area and excellent chemical and physical stability [11]. Resin of moderate to high porosity increases the accessibility of the ligands as well as the metal ions for the chelating site. Selective chelating resin with high metal ion uptake capacity may be designed by immobilizing a small sized polydentate ligand moiety onto Amberlite XAD resins either through surface sorption or chemical modification. Chemical modification involves the insertion of an appropriate functional group (linkage/spacer group) such as -N=N-, -CH2-, -N=C- on the surface of polymeric support and then immobilization of a particular ligand by a condensation reaction or coupling reaction. Chelating resin prepared by chemical linkage exhibits better resistance to the leaching of the ligands. High selectivity may be attributed to their function as chelate formation, ion exchange and physical sorption. Salicylic acid (SA) is a ligand with a carboxylic and a phenolic functional group which forms chelates with a number of metal ions [12-14]. It has already been used, for example, for the spectrophotometric determination of copper [15], aluminum [16,17], and iron [18]. Certain chelating resins like Amberlite XAD- 2-salicylic acid [19], Amberlite XAD-4-salicylic acid (grafting via a ketone bridge) [20], Silica gel- salicylic acid [21] have been synthesized and characterized but their applications with analytical figures of merit have not been explored. Therefore, it was thought worthwhile to functionalize Amberlite XAD-4 with salicylic acid by coupling through azo spacer (Figure 3.1). The chelating resin was used for the preconcentration of Cu(II), Cr(III), Zn(II), Cd(II), Mn(II), Ni(II) and Co(II) from 93 environmental and various other real matrices prior to their determination by FAAS and was found to have superior preconcentration and sorption characteristics for metal ions compared to other salicylic acid functionalized chelating resins. The proposed method was validated by analyzing standard reference materials (both environmental and biological) and by performing recovery studies. The proposed method was validated by analyzing environmental and biological standard reference material.

-CH-CH2-

N N

OH O

n+ a M Figure 3.1 Structure of a monomeric unit of AXAD-4 modified with Salicylic acid (SA); ‘a’ is the probable chelating sites.

3.2 Experimental

Both batch and column methods (Section 2.6) have been employed for the sorption and elution studies of Cu(II), Cr(III), Zn(II), Cd(II), Mn(II), Ni(II) and Co(II) prior to their determination by flame atomic absorption spectrometry (FAAS). The recommended procedures (Section 2.5-2.6) have been applied for the determination of different experimental parameters, including physico-chemical properties, of the resin.

The physico-chemical properties that have been determined include:

 Water regain capacity of the modified resin was determined by the recommended procedure as described in section 2.5.1 94

 Hydrogen ion capacity of the modified resin has also been evaluated (Section 2.5.2)

The experimental parameters that have been optimized include:

 Optimum pH range for sorption was ascertained by applying the recommended procedure (Section 2.6.1)  Contact /half-loading time for sorption has been determined (Section 2.6.1)  Flow rate for sorption as well as for elution has also been determined (Section 2.6.2)  Eluting agent for complete desorption (Section 2.6.3)  The recommended procedure (Section 2.6.2) was followed to determine the preconcentration factor  Breakthrough volume for sorption has been determined according to the recommended procedure (Section 2.6.2)  The recommended procedure (Section 2.6.2) was followed for the determination of limit of detection (LOD) for each metal.

The following experiments have been performed for the validation of the method:

 Recovery of metal ions from standard reference materials (Section 2.7)  Recovery of metal ions from standard metal ion solutions has been ascertained (Section 2.7)  Student’s t-test has been performed (Section 2.7)

The method has been applied for the following applications:

3.3 Results and discussion

3.3.1 Characterization of AXAD-4-SA

In order to study the extent of product formation, elemental analysis was carried out

at each stage of the reaction. The nitrogen content of the nitrated resin and the subsequent

reduced product was found to be 9.38% (6.71 mmol g–1) and 11.76% (8.40 mmol g–1) respectively. Elemental analysis of AXAD-4-SA gave C 63.36%, H 4.56%, and N 10.12% which are in agreement with calculated values for C15H12N2O3·H2O as %C 62.48, %H 4.93

and %N 9.77.

The resin was subsequently characterized by IR spectral data. The FT-IR spectrum of

AXAD-4-SA (Figure 3.2) has prominent bands at 1680cm-1, 1645cm-1, 1486cm-1, and

1387cm-1 due to carboxylate, -N=N-, OH (bending), and phenolic group vibrations,

respectively. This supports the immobilizing of SA onto Amberlite XAD-4 resin. The absence of broadening of the hydroxyl band in the spectra of the metal loaded resin proves the absence of hydrogen bonding that indicates the participation of –OH group in the coordination process. The red shifts of the two peaks namely hydroxyl and carboxylic by 10-

15 cm-1 for metal loaded resin further suggest that chelation with salicylic acid functionality

is responsible for the sorption of metal ions by AXAD-4 resin, whereas the –N=N- band

remained unchanged. The resin shows good chemical stability with no loss of capacity up to

-1 5 mol L of HCl/HNO3/H2SO4 used for stripping of metal ions. It can withstand alkaline medium up to 4 mol L-1 NaOH. At concentrations higher than 4 mol L-1 of NaOH, the sorption capacity gets reduced by 3.5%. According to thermogravimetric analysis (Figure

3.3), the resin was found to be stable up to 200°C with no significant loss of weight other than the loss due to sorbed water (5.72%). Weight loss of 14.47% up to 382.71°C in TGA was supported by an endothermic peak in the DTA curve indicate the loss of functional group due to the degradation of SA reagent in the chelating resin.

Figure 3.2 FT-IR spectrum of a) AXAD-4-SA and b) AXAD-4-SA saturated with Cu(II)

Figure 3.3 TGA/DTA curves of AXAD-4-SA

The water regain capacity was found to be 11.45 mmol g-1. This value reflects the high hydrophilicity of the resin which is satisfactory for column operation. The overall hydrogen ion capacity was found to 7.15 mmol g-1 of resin, which may be contributed by the two replaceable hydrogen ions of the hydroxyl and the carboxylic moieties. Theoretically, if 3.7 mmol of the reagent constituted per gram of the resin, the hydrogen ion capacity, due to the hydroxyl and carboxylic groups should have been 7.40 mmol g-1, which agrees with the experimental value.

3.3.2 Optimum experimental parameters In order to optimize sorption of metal ions, the multivariate approach was followed to establish all the parameters. Each optimum condition was established by varying one of them and following the recommended procedure.

Figure 3.4 Dependence of sorption capacity on the pH of the solution (Batch method parameter: 50 mL, solution; 100 µg mL-1, metal ions; 0.2 g, resin)

Effect of pH for metal ion uptake Optimum pH of metal ion uptake was determined by static method. Excess of each metal ion (50 mL, 100 µg mL-1) was shaken with 0.2 g of resin for 120 minutes. The pH of 98

metal ion solution was adjusted prior to equilibration over a range of pH 2-10 (±0.01) with the corresponding buffer system. The pH > 10 was not studied to avoid precipitation due to the formation of metal hydroxide. The effect of pH on the sorption of metal ions on AXAD- 4-SA is shown in Figure 3.4. As the complex formation is strongly pH dependent, careful adjustment of proper pH for the reagent was necessary. All the metal ions studied exhibited higher sorption capacity at pH 5.5-8.0 (±0.01) except for Cu(II) which shows high sorption at pH 10±0.01. Hence, pH 5.57±0.01 was adjusted in all further experiments for all metals except Cu(II) which was adjusted at pH 10±0.01.

Figure 3.5 Kinetics of sorption of metal ions on AXAD-4-SA (Batch method parameter: 50 mL, solution; 100 µg mL-1, metal ions; 0.2 g, resin)

Sorption kinetics and loading halftime The rate of loading of metal ions on the resin was determined by static method. 50 mL of each metal ion solution (100 µg mL-1) was shaken with 0.2 g of the resin in a

thermostat shaker for pre-selected intervals of time. The loading halftime, t1/2, that is, the

time required to reach 50% of the resins total loading capacity was evaluated from the resulting isotherm. From the kinetics of sorption for each metal (Figure 3.5), it was observed that 50 min was enough for the sorbent to reach the saturation level for all the metals. The sorption rate constant k can be calculated using the following equation: -ln (1-F) = kt, where

F = Qt/Q and Qt is the sorption amount at sorption time t and Q the sorption amount at

equilibrium. Putting the value of Qt at t1/2 in the above equation we may get the corresponding value of k for every metal ion (Table 3.1).

Table 3.1 Kinetics and batch capacity of sorption of metal ions on AXAD-4-SA (Batch method parameter: 50 mL, solution; 0.2 g, resin) Loading halftime Rate constant Batch capacity Metal ion -1 -2 -1 t1/2 (min) k (min. ) ×10 (µmol g ) Cu(II) 10 6.93 245.0 Cr(III) 10 6.93 156.2 Zn(II) 15 4.62 155.0 Cd(II) 15 4.62 145.0 Mn(II) 12 5.77 125.0 Ni(II) 12 5.77 122.5 Co(II) 12 5.77 70.0

Effect of resin amount on the sorption capacity To investigate the effect of the amount of the resin on the sorption of metal ions, an excess of the metal ion solution corresponding to 150 µg mL-1 was equilibrated with varying amounts of resin buffered at the suitable pH. The retention of the metal ions per gram of the resin increased with the increase in the amount of the resin. An almost constant and maximum sorption capacity was observed after 0.2 g of the resin.

Effect of flow rate for sorption and elution The flow rate for the sorption of metal ions was changed within 1-10 mL min−1, and the results showed that the flow rate has no effect on the sorption of metal ions up to 4 mL min−1, and in the higher flow rates (>4 mL min−1) the sorption of metal ions decreased at

100

least 20%. The eluent flow rate had no effect on the stripping of metal ions up to 3 mL min−1, and the recovery was quantitative. Hence, such fast flow rate makes it superior to the previously reported works employing salicylic acid as the chelating ligand [19].

Types of eluting agents In order to investigate the most efficient eluting agent, varying concentration of

different volumes of HNO3, HCl and H2SO4 were tried. Other acids such as formic acid, acetic acid and perchloric acid were also investigated. Distilled water was found to be unsuitable for the purpose of elution as <0.5% recovery was achieved indicating that the metal ions were retained by the resin by some strong bonding forces. Among the mineral acids, 4 mol L-1 HCl was found to give >98% recovery of Cu(II), Ni(II), Cd(II) and Mn(II) -1 with 5 mL, respectively while 4 mol L HNO3 could give >99% recovery of Zn(II), Co(II) and Cr(III). Table 3.2 depicts the results.

Table 3.2 Elution of metal ions from the AXAD-4-SA (Column parameter: 3 mL min-1, elution flow rate; 0.2 g, resin) Stripping agent Mean recovery for five replicates (%) Concentration Volume Type Cu(II) Cr(III) Zn(II) Cd(II) Mn(II) Ni(II) Co(II) mol L-1 (mL) 2 5 25 15 18 41 70 28 25 HCl 4 5 99 29 52 96 99 92 40 1 5 21 32 17 33 45 25 51 H2SO4 2 5 24 36 26 40 48 35 58 1 5 56 42 29 36 24 51 38

HNO3 2 5 88 48 34 62 32 97 61 4 5 39 98 98 69 35 95 97 1 5 41 34 29 35 25 38 40 HCOOH 2 10 54 40 33 39 28 43 49 2 5 10 8 13 9 21 18 25 CH3COOH 4 10 12 14 13 10 25 18 26 2 5 34 24 38 20 16 28 41 HClO4 4 10 42 27 39 26 18 28 48

101

Resin reusability test For the reusability of the resin, the loading solution of the metal ions taken several times on a column having a concentration of 50 µg mL-1 at a flow rate of 3 mL min-1 and eluting by the appropriate eluting agent. The capacity of the modified resin was found to be apparently constant (less than 3%) after the repeated use of more than 35 cycles of sorption and desorption as shown in Figure 3.6.

Figure 3.6 Regenerability of AXAD-4-SA for different metals (Column parameters: 3 mL min-1, elution flow rate; 50 µg mL-1, metal ions; 0.2 g, resin)

Study of interferences Various cations and anions, which are inevitably associated with heavy metals, may interfere in the latter’s determination through precipitate formation, redox reactions, or competing complexation reactions. In order to assess the analytical applicability of the resin to real samples, common chemical species such as sodium citrate, sodium tartrate, sodium - 2- + 2- 3- – + + oxalate, humic acid, fulvic acid, NO3 , CO3 , NH4 , SO4 , PO4 , Cl , K and Na were checked for any interference in the sorption of these metals. Very few literatures [22, 23]

102

have considered the interference of these humic substances which are generally present in natural waters at µg mL-1 to ng mL-1 levels and form complexes with various heavy metals [24-26]. The tolerance limit is defined here as the species concentration causing a relative error smaller than ±5% related to the preconcentration and determination of the analytes. The tolerance limit for each metal (Table 3.3) is found to be much higher than that reported in literature [19].

Table 3.3 Tolerance limit of foreign species (in binary mixtures) on sorption of metal ions (Column parameter: 50 mL, solution; 5 µg, metal ion; 0.2 g, resin) Foreign species Tolerance limit of metal ions (µg mL-1)

Cu(II) Cr(III) Zn(II) Cd(II) Mn(II) Ni(II) Co(II)

NaCl 20000 15000 20000 20000 20000 20000 20000

Na2SO4 10000 10000 10000 10000 10000 10000 10000

NaNO3 15000 10000 15000 10000 15000 15000 10000

Na2PO4 500 300 500 250 400 400 400

NH4Cl 20000 15000 15000 10000 15000 15000 15000

Sodium citrate 250 300 300 300 250 400 350

Sodium oxalate 85 80 70 100 80 100 100

Sodium potassium tartrate 65 50 100 150 120 90 100

CH3COONa 10000 8000 10000 7000 6000 12000 10000

CaCl2 20000 10000 15000 10000 10000 20000 20000

MgCl2 20000 10000 15000 15000 15000 20000 20000

Humic acid 40 55 60 60 65 65 65

Fulvic acid 35 40 45 45 50 50 45

Adsorption isotherm In order to determine the sorption capacity of the resin, test solutions of Cu(II), Cr(III), Zn(II), Cd(II), Mn(II), Ni(II) and Co(II) weighing in the range 100-50,000 µg were

103

taken in batches containing 0.2 g of chelated resin and recoveries were investigated. Langmuir isotherms were plotted in order to determine the resin capacity in accordance to the literature [27]. The Langmuir adsorption isotherm is described by the equation given as,

Ce/qe = 1/qmb + Ce/qm,

−1 where qe is the amount of metal sorbed per unit weight of the resin (mg g ) at

−1 equilibrium, Ce is the final concentration in the solution (mg L ), qm is the maximum

sorption at monolayer coverage (mg g−1), and b is the sorption equilibrium constant, which is

related to the energy of sorption. A plot of Ce/qe versus Ce shows linearity and hence, the

Langmuir constants, qm and b, can be calculated from the slope and intercept of the plot (Table 3.4).

Table 3.4 Langmuir isotherm constants for sorption of metal ions (Column parameters: 4.0 mL min-1, sorption flow rate; 0.2 g, resin). b qm Standard Metal ions R2 (L mg-1) (mg g-1) deviation (N=5) Cu(II) 0.0327 15.36 0.9989 0.3622 Cr(III) 0.0539 8.01 0.9992 0.2310 Zn(II) 0.0515 10.16 0.9997 0.3016 Cd(II) 0.0870 16.12 0.9983 0.4324 Mn(II) 0.0153 6.66 0.9986 0.2118 Ni(II) 0.0191 7.02 0.9999 0.1948 Co(II) 0.0152 4.12 0.9982 0.3133

From the plots obtained for each metal, the amount of maximum total metal (qm) sorbed on 1.0 g of resin is calculated. The maximum sorption capacity calculated from the Langmuir isotherm (Figure 3.7) indicated that the resin retained Cu(II) more strongly than the other metal ions under study.

104

Figure 3.7 Langmuir sorption isotherms depicting the sorption behaviors of metal ions onto AXAD-4-SA (Column parameters: 4 mL min-1, sorption flow rate; 0.2 g, resin)

The higher surface area of AXAD-4 resin complemented by highly efficient incorporation of SA may be the contributing factors for higher sorption. This fact is also supported by the high hydrophilicity of AXAD-4-SA which facilitates better surface contacts with the metal ions in aqueous solutions.

Preconcentration factor and breakthrough capacity The limit of preconcentration was determined by increasing the volume of metal ion solution and keeping the total amount of loaded metal ion constant at 10 µg. The breakthrough volume corresponds to the volume at which the effluent concentration of metal ions from the column is about 3–5% of the influent concentration. The breakthrough volume was determined by the dynamic procedure. The overall capacity, breakthrough capacity and the degree of utilization were also determined [28]. Figure 3.8 gives the breakthrough curves for all the metal studied. 105

Figure 3.8 Breakthrough curves for sorption of metal ions: C/Co is the concentration ratio of the effluent to influent (Column parameters: 4 mL min-1, sorption flow rate; 0.5 g, resin)

The overall sorption capacity calculated on the basis of total saturation volume was

compared with the corresponding breakthrough capacities for each metal. The closeness of

the dynamic capacity to the total sorption capacity reflects the applicability of the column

technique for preconcentration. Quantitative collection of metal ions was possible from

solutions of concentration in the order of 5.5-10.0 µg mL-1 with a recovery up to 98%

resulting in a preconcentration factor of 200-360 (Table 3.5). The preconcentration factor of

Zn is higher than previously reported literature [19].

106

Table 3.5 Preconcentration and breakthrough profiles of metal ions on AXAD-4-SA (Column parameter: 4 mL min-1, sorption flow rate; 3 mL min-1, elution flow rate; 0.5 g, resin) Preconcentration studies Breakthrough studies Overall Metal Total Concentration Precon- Breakthrough Breakthrough Degree of sorption ions volume limit centration capacity capacity volume column (mL) (ng mL-1) factor (µmol (µmol g-1) (mL) utilization g-1) Cu(II) 1800 5.50 360 245.0 214.3 1700 0.87 Cr(II) 1600 6.25 320 156.2 123.1 1500 0.78 Zn(II) 1600 6.25 320 155.0 122.0 1500 0.78 Cd(II) 1500 6.45 300 145.0 119.5 1400 0.82 Mn(II) 1400 7.14 280 125.0 110.0 1300 0.88 Ni(II) 1400 7.14 280 122.5 106.2 1300 0.86 Co(II) 1000 10.00 200 70.0 60.1 950 0.85

3.4 Method validation

The accuracy of the present method was evaluated from the results of the analysis of various Standard Reference Materials (SRMs) including environmental, biological and alloy samples applying recommended column procedure. Calculated Student’s t (t-test) values for respective metal ions were found to be less than the critical Student’s t-value of 2.78 at 95% confidence level for N=5 (Table 3.6). Hence, the mean values were not statistically significant from the certified values indicating that the method could be applied successfully for the analysis of real samples constituting different matrices.

Using optimum conditions, the precision of the method was evaluated. Six successive sorption and elution cycles of 10 µg of each metal ion taken in 100 mL (eluted in 5 mL of 4 -1 mol L HCl/HNO3) were performed following the recommended procedure. It was found that the mean percentage recoveries of all the metal ions studied were 97.8-100.7% at 95 % confidence level. The RSD values were calculated to be below 5 %. The results of water analysis with RSD < 5 % support the applicability of the method.

107

Table 3.6 Analysis of metal ions in standard reference materials (Column parameter: 50 mL, solution; 4 mL min-1, sorption flow rate; 3 mL min-1, elution flow rate, 0.2 g, resin) Calculated Certified value Found by proposed method Samples Student's ‘t’ (µg g-1) µg g-1 (RSD )a value b Vehicle exhaust Cd:1.1, Co:3.3, Cu: Cd:1.06 (4.3), Co:3.2 (2.8), 1.96, 2.49, particulates 67, Ni: 18.5 Cu:64.5 (3.4), Ni: 17.8 (3.2) 2.54, 2.74 NIES 8 c Human hair Mn:5.2, Zn:169, Mn:5.0 (4.0), Zn:163.8 (2.9), 2.23, 2.44, NIES 5 Cu:16.3, Ni:1.8, Cu: 15.9 (2.8), Ni: 1.7 (4.8) 2.00, 2.74 Tea leaves Mn:700, Zn:33, Mn:690.0 (1.9), Zn:31.6 (3.8), 1.70, 2.60, NIES 7 Cu:7.0, Ni:6.5 Cu:6.7 (4.5), Ni:6.2 (4.3) 2.22, 2.51 Rompin Mn:2200, Cu:640, Mn:2185.5 (1.1), 1.34, hematite, Zn: 1030 Cu:630.2 (1.9), Zn:1019.4 (1.4) 1.83, 1.66 JSS (800-3) d Zinc base die- Cu:1320, Cd:51, Cu:1310.2(1.2), Cd:49.1(4.1), 1.39, 2.11, casting alloy C Mn: 140, Ni: 29 Mn: 134.9(3.5), Ni: 27.5(4.6) 2.41, 2.65 NBS 627 e a Relative standard deviation, n = 5 ; b at 95 % confidence level; c National Institute of Environmental Studies (NIES); d Iron and Steel

Institute of Japan (JSS); e National Bureau of Standards (NBS)

A blank run was performed applying recommended column procedure with 100 mL of aqueous solution prepared by adding suitable buffer (excluding metal ions) and finally eluting the same in 5 mL before subjecting it to FAAS determination. The detection limits, evaluated as three times the standard deviation (s) of the blank signal, were found to be 0.42, 0.57, 0.63, 0.77, 0.94, 0.96 and 1.41 µg L-1 for Cu(II), Cr(III), Zn(II), Cd(II), Mn(II), Ni(II) and Co(II) respectively.

3.5 Applications

Applicability of the present method for preconcentration and determination of metal ions was accomplished by analyzing 500 mL of each water sample following recommended

108 column procedure (direct method). The metal ion determinations were also confirmed using the method of standard additions from various real water (500 mL) samples which were spiked with known amount (5 µg) of individual metal ions. Recommended column procedure was then applied to determine the total metal ion contents (Standard Additions Method, S.A). The close agreement of the results found by direct with that found by standard addition method (Table 3.7) indicates the reliability of the present method for metal analyses in water samples of various matrices without significant interference.

Table 3.7 Determination of metal ions in natural water after preconcentration by AXAD-4-SA column (Column parameter: 500 mL, solution; 4 mL min-1, sorption flow rate; 3 mL min- 1,elution flow rate, amount of resin 0.5 g) Metal ion found by proposed method µg L-1 (±confidence limita ) Samples Method Cu(II) Cr(III) Zn(II) Cd(II) Mn(II) Ni(II)

Canal Direct 15.2±0.90 5.7±0.58 7.2±0.33 N.D.b 3.6±0.27 6.4±0.36 water S.Ac 15.7±0.81 6.1±0.59 7.5±0.46 1.2±0.12 3.5±0.27 6.5±0.38

Tap Direct 11.9±0.85 9.7±0.69 15.4±1.18 N.D.b 10.3±0.74 5.8±0.31 water S.A. 12.1±0.99 10.2±0.83 16.3±1.29 1.1±0.12 10.4±0.80 5.9±0.35

Sewage Direct 10.2±1.16 6.5±0.66 4.1±0.26 4.6±0.20 5.1±0.29 11.8±0.87 water S.A. 10.5±1.01 6.8±0.64 4.5±0.26 4.6±0.21 5.2 ±0.28 11.9±0.94

River Direct 19.6±1.80 4.9±0.25 6.9±0.56 2.8±0.16 6.6±0.27 4.4±0.27 water S.A. 20.4±1.11 5.3±0.25 7.5±0.57 2.9±0.15 6.8±0.33 4.5±0.31 ts a Confidence limit, C.L = x ± , n=3 at 95% confidence level; b N.D.= not detected; c S.A.=Standard addition. N

The proposed method has also been applied for the determination of metal ions in multivitamin formulation and food samples (IMS and vanaspati ghee). The observed results obtained were found to be accurate and in close agreement with the reported value (Table 3.8).

109

Table 3.8 Determination of metal ions in capsule and food samples (Column parameter: 50 mL, solution; 4 mL min-1, sorption flow rate; 0.2 g, resin) Reported value Found by proposed method Samples (µg g-1) µg g-1 (RSD )a

Maxirich (Cipla) Cu:398.2; Zn:442.5 Cu:390.3 (2.7), Zn:440.6 (3.3) Lactogen 1(Nestle) Cu:2.9; Zn:37.0 Cu:2.8 (1.9), Zn:35.8 (2.5) Vanaspati ghee Ni:0.45 Ni:0.40 (2.3) a Average of five determinations

3.6 Conclusion

The chelating ability of SA has been utilized in developing chelating sorbents for the purpose of separation and preconcentration of trace metal ions. The results reflect its promising nature for trace metal ion analysis in various natural water resources, environmental and biological samples. The main advantages of this procedure are the simple and fast preparation of the chelating resin and no requirement of organic solvents in the metal elution step. The excellent ability for the exclusion of alkali and alkaline earth elements on the AXAD-4-SA resin makes it desirable for use in the separation and preconcentration of trace elements because their presence often interfere in the subsequent FAAS determination. The results obtained demonstrated good reproducibility. Moreover, the use of a column preconcentration technique allows for the assessment of low trace metal concentrations, even by less sensitive determination methods such as FAAS. As compared to other previous works [19-21] employing chelating resins using salicylic acid as the chelating ligand, the present work covers more experimental parameters (such as breakthrough volume, Langmuir isotherm, matrices (including humic and fulvic acids) for interference studies, and has been found to be superior in various aspects, such as flow rate, preconcentration factor, half- loading time and sorption capacity. The present work also reports lower LOD (Limit of detection) that makes it more feasible for the preconcentration and determination of trace metal ions. Preconcentration by this material (AXAD-4-SA) from river water samples do not require any prior digestion of the samples. It can be successfully applied for the analysis of both environmental and biological samples as indicated by the high precision and absence of systematic errors. 110

References

[1] T.P. Rao, R.S. Praveen, S. Daniel, Styrene-divinyl benzene copolymers: synthesis, characterization, and their role in inorganic trace analysis, Crit. Rev. Anal. Chem. 34 (2004) 177.

[2] S. Nabi, A. Alim, A. Islam, M.Amjad, Column chromatographic separation of metal ions on 1-(2-pyridylazo)-2-napthol modified Amberlite IR-120 resin, J. Sep. Sci. 28 (2005) 2463.

[3] S. Dutta, A.K. Das, Synthesis, Characterization, and Application of a New Chelating Resin Functionalized with Dithiooxamide J Appl Polymer Sci, 103 (2007) 2281.

[4] S. Xingguang, W. Meijia, Z. Yihua, Z. Jiahua, Z. Hanqi, J. Qinhan, Semi-online preconcentration of Cd, Mn and Pb on activated carbon for GFASS, Talanta 59 (2003) 989.

[5] H. Bag, A.R. Turker, R. Coskun, M. Sacak, M. Yigitoglu, Determination of zinc, cadmium, cobalt and nickel by flame atomic absorption spectrometry after preconcentration by poly(ethylene terephthalate) fibers grafted with methacrylic acid, Spectrochim. Acta B 55 (2000) 1101.

[6] E. Kenduzler, A.R. Turker, Atomic absorption spectrophotometric determination of trace copper in waters, aluminium foil and tea samples after preconcentration with 1-nitroso-2- naphthol-3,6-disulfonic acid on Ambersorb 572, Anal. Chim. Acta 480 (2003) 259.

[7] S.A. Nabi, Alimuddin, A. Islam, Synthesis and characterization of a new cation exchanger-zirconium(IV)iodotungstate: Separation and determination of metal ion contents of synthetic mixtures, pharmaceutical preparations and standard reference material, J. Hazard. Mater. 172 (2009) 202.

[8] N. Rajesh, B. Deepthi, A. Subramaniam, Solid phase extraction of chromium(VI) from aqueous solutions by adsorption of its ion-association complex with acetyltrimethyl- ammoniumbromide on an alumina column, J. Hazard. Mater. 144 (2007) 464.

[9] C. Ekinci, U. Koklu, Determination of vanadium, manganese, silver and lead by graphite furnace atomic absorption spectrometry after preconcentration on silica-gel modified with 3-aminopropyltriethoxysilane, Spectrochim. Acta B 55 (2000) 1491.

[10] H.A. Mottoland, J.R. Steimetz, Chemically Modified Surfaces, Amsterdam, Elsevier, 1992.

[11] WANG, Hai-ling; FEI, Zheng-hao; CHEN, Jin-long; ZHANG, Quan-xing; XU, Yan-hua. Application of multiwalled carbon nanotubes treated by potassium permanganate for determination of trace cadmium prior to flame atomic absorption spectrometry, J Environ Sci 19 (2007) 1298.

[12] K.E. Jabalpurwala, K.A. Venkatachalam, M.B. Kabadi, Metal-ligand stability constants of some ortho-substituted phenols, J. Inorg. Nucl. Chem. 26 (1964) 1027. 111

[13] D.D. Perrin, L.G. Sillen, Stability Constants of Metal-Ion Complexes, Part B: Organic Ligands; Pergamon Press: Oxford, New York, 1979.

[14] L.G. Sillen, A.E. Martell, J. Bjerrum. Stability Constants of Metal-Ion Complexes; Chemical Society: London, 1964.

[15] A. Saha, K. Baksi, Spectrophotometric of salicylic acid in pharmaceutical formulations using copper(II) acetate as a colour, Analyst 110 (1985) 739.

[16] E. Rakotonarivo, C. Tondre, J.Y. Bottero, J. Mallevialle, Polymerized and hydrolyzed aluminium(III) complexation by salicylate ions. Kinetics and thermodynamic study, Water Res. 23 (1989) 1137.

[17] C.C. Ainsworth, D.M. Freidrich, P.L. Gassman, Z. Wang, A.G. Joly, Characterization of salicylate-alumina surface complexes by polarized fluorescence spectroscopy, Geochim. Cosmochim. Ac. 62 (1998) 595.

[18] K.W. Cha, K.W. Park, Determination of iron(III) with salicylic acid by the fluorescence quenching method, Talanta 46 (1998) 1567.

[19] R. Saxena, A.K. Singh, D.P.S. Rathore, Salicylic acid functionalized polystyrene sorbent Amberlite XAD-2. Synthesis and applications as a preconcentrator in the determination of Zinc(II) and Lead(II) by Using Atomic Absorption Spectrometry, Analyst 120 (1995) 403.

[20] S. Boussetta, C. Branger, A. Margaillan, J.L. Boudenne, B. Coulomb, Salicylic acid and derivatives anchored on poly(styrene-co-divinylbenzene) resin and membrane via a diazo bridge: Synthesis, characterization and application to metal extraction, React. Funct. Polym. 68 (2008) 775.

[21] Q. Xu, P. Yin, G. Zhao, G. Yin, R. Qu, Synthesis and characterization of silica gel microspheres encapsulated by salicyclic acid functionalized polystyrene and its adsorption of transition metal ions from aqueous solutions, Cent. Eur. J. Chem. 8 (2009) 214.

[22] M.D.G. Castro, M.D.G. Riano, M.G. Vargas, Separation and preconcentration of cadmium ions in natural water using a liquid membrane system with 2-acetylpyridine benzoylhydrazone as carrier by flame atomic absorption spectrometry, Spectrochim. Acta B 59 (2004) 577.

[23] Y. Liu, Y. Guo, S. Meng, X. Chang, Online separation and preconcentration of trace heavy metals with 2,6-dihydroxyphenyl-diazoaminoazobenzene impregnated amberlite XAD-4, Microchim. Acta 158 (2007) 239.

[24] W.S. Gardner, P.F. Landrum, Characterization of ambient levels of ultraviolet-absorbing dissolved humic materials in natural waters by aqueous liquid chromatography, In: Christman RF, Gjessing ET (Eds) Aquatic and Terrestrial Humic Materials, Ann Arbor Science, Ann Arbor, MI, 1983, pp. 203.

112

[25] J. Buffle, R.S. Altmann, M. Filella, Special issue on humic and fulvic compounds, Anal. Chim. Acta 232 (1990) 3.

[26] S.D. Khattri, M.K. Singh, Sorption, recovery of metal ions from aqueous solution using humus, Indian. J. Chem. Tech. 3 (1999) 114.

[27] A.W. Adamson, Physical Chemistry of surfaces, Wiley-Interscience, New York, 1990

[28] F. Helfferich, Ion Exchange, McGraw Hill, New york, 1962, p.424.

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Chapter 4

Preparation, Characterization of a Novel Chelating Resin Functionalized with o-Hydroxybenzamide and Its Application for Preconcentration of Trace Metal Ions 4.1 Introduction

Solid phase extraction using chelating resin for the preconcentration and separation of metal ions has been preferred over conventional solvent extraction and coprecipitation [1-7]. The possibility to extract selectively a number of analytes over a wide pH range, quantitative sorption and elution, kinetically faster sorption and desorption mechanisms, good retention capacity, high preconcentration factor and regeneration of resins over many cycles with good reproducibility in the sorption characteristics are frequently quoted as an advantage [8].

Complexing sorbents destined for preconcentration of heavy metals are synthesized in different ways: the chemical modification of polymeric and mineral matrices (grafting of functional groups) or the non-covalent immobilization of reagents (ligands) on various supports [9-12]. In fact, the mechanism of the interaction of metals with complexing sorbents is complicated, and depends on both the type of complex-forming groups and the sorption conditions [13-15].

Amberlite XAD resins, as the copolymer backbone for the immobilization of chelating ligands, have some physical superiority, such as porosity, uniform pore size distribution, high surface area and chemical stability towards acids, bases, and oxidizing agents, as compared to other resins. Among these adsorbents, Amberlite XAD resins (XAD- 2, XAD-4, XAD-7, XAD-16, XAD-1180 and XAD-2000) are very useful for the preconcentration of metal complexes [16-19].

Therefore, it was thought worthwhile to functionalize Amberlite XAD-4 (AXAD-4) with o-hydroxybenzamide (HBAM) (Figure 4.1) so as to develop a method for the preconcentration and separation of trace metal ions from real samples constituting different matrices. To our literature knowledge, no study is available on the use of Amberlite XAD-4 fixed with o-hydroxybenzamide as sorbent for the preconcentration of trace metal ions from various media. Various parameters influencing the quantitative recoveries of analytes, including pH of sample, sample volume, eluent volume and effect of foreign ions have been studied. The proposed method was successfully applied to the determination of trace metals in water samples, fish, urine, multivitamin tablets, infant milk substitute, and SRM samples.

114

-CH-CH2-

N N

NH2

OH O

n+ a M Figure 4.1 Structure of a monomeric unit of AXAD-4 modified with o- Hydroxybenzamide (HBAM); ‘a’ is the probable chelating sites

4.2 Experimental

Both batch and column methods (Section 2.6) have been employed for the sorption and elution studies of Zn(II), Co(II), Ni(II), Cu(II) and Pb(II) prior to their determination by flame atomic absorption spectrometry (FAAS). The recommended procedures (Section 2.5-2.6) have been applied for the determination of different experimental parameters, including physico-chemical properties, of the resin.

The physico-chemical properties that have been determined include:

 Water regain capacity of the modified resin was determined by the recommended procedure as described in section 2.5.1  Hydrogen ion capacity of the modified resin has also been evaluated (Section 2.5.2)

The experimental parameters that have been optimized include:

 Optimum pH range for sorption was ascertained by applying the recommended procedure (Section 2.6.1)  Contact /half-loading time for sorption has been determined (Section 2.6.1)

115

 Flow rate for sorption as well as for elution has also been determined (Section 2.6.3)  Eluting agent for complete desorption (Section 2.6.3)  The recommended procedure (Section 2.6.2) was followed to determine the preconcentration factor  Breakthrough volume for sorption has been determined according to the recommended procedure (Section 2.6.2)  The recommended procedure (Section 2.6.2) was followed for the determination of limit of detection (LOD) for each metal.

The following experiments have been performed for the validation of the method:

The method has been applied for the following applications:

 Collection and pretreatment (Section 2.3.1) of natural waters prior to determination of trace metal ions.  Determination of Cu(II) and Zn(II) in multivitamin formulation and infant milk substitute was carried out after digestion of the samples according to the recommended procedure as in section 2.3.2.  Digestion of standard reference materials were carried out according to the recommended procedure as in section 2.3.3.  The recommended procedure (Section 2.3.4) was followed for the digestion of fish samples prior to their analysis for metal content.  Collection and pretreatment (Section 2.3.4) of urine sample prior to determination of trace metal ions.

4.3 Results and discussion

116

4.3.1 Characterization of AXAD-4-HBAM In order to study the extent of product formation, elemental analysis was carried out at each stage of the reaction scheme.

Figure 4.2 FT-IR spectrum of a) AXAD-4-HBAM and b) AXAD-4- HBAM saturated with Cu(II)

The nitrogen content of the nitrated resin and the subsequent reduced product was found to be 9.38% (6.71 mmol g–1) and 11.76% (8.40 mmol g–1) respectively. Elemental analysis of AXAD-4-HBAM gave C 67.16%, H 5.59%, and N 15.67% which are in agreement with calculated values for C15H14N3O2·H2O as %C 66.08, %H 5.72 and %N 15.12. The IR spectrum of the modified resin AXAD-4-HBAM (Figure 4.2) was compared with that of untreated Amberlite XAD-4. The band at 3490 cm-1 may be assigned to the phenolic hydroxyl group, which suggests the presence of intramolecular hydrogen bonding. While the other four additional bands at 1707, 1608, 1557 and 1370 cm-1, which may be contributed to carbonyl (> C=O), azo (–N=N–) stretching vibrations besides -N-H, and –C- N- (bending), respectively. The IR spectrum of the metal ion-free chelating resin was compared with those of the metal ion-saturated resin. A red shift (2–5 cm-1) in the bands of

117 the carbonyl group and the disappearance of the phenolic hydroxyl group in the metal ion- saturated AXAD-4-HBAM suggest that chelation of metal ions through –OH and carbonyl (> C=O) groups is probably responsible for metal sorption.

Figure 4.3 TGA/DTA curves of AXAD-4-HBAM

The resin shows good chemical stability with no loss of capacity up to 4 mol L-1 of

HCl/HNO3/H2SO4 used for stripping of metal ions. It can withstand alkaline medium up to 4 mol L-1 NaOH. At concentrations higher than 4 mol L-1 of NaOH, the sorption capacity gets reduced by 3.5%. According to thermogravimetric analysis (Figure 4.3), the resin was found to be stable up to 200°C with no significant loss of weight other than the loss due to sorbed water (6.51%). Weight loss of 18.88% up to 306.60°C in TGA was supported by an endothermic peak in the DTA curve indicate the loss of functional group due to the degradation of HBAM reagent in the chelating resin. The overall hydrogen ion capacity was found to be 7.68 mmol g-1 which is contributed by the two replaceable hydrogen ions of the hydroxyl and the carboxylic moieties. The water regain capacity was found to be 12.93 mmol g-1. 118

4.3.2 Optimum experimental parameters In order to optimize sorption of metal ions, the univariate approach was followed to establish all the parameters. Each optimum condition was established by varying one of them and following the recommended procedure.

Effect of pH for metal ion uptake The optimum pH of metal ion uptake was determined by a static method. An excess of each metal ion (50 mL, 100 µg mL-1) was shaken with 0.2 g of resin for 120 minutes. The pH of the metal ion solution was adjusted prior to equilibration over a range of pH 2-10 (±0.01) with the corresponding buffer system. The effect of pH on the sorption of metal ions on AXAD-4-HBAM is shown in Figure 4.4.

Figure 4.4 Dependence of sorption capacity on the pH of the solution (Experimental conditions: 50 mL, solution; 100 µg mL-1, metal ions; 0.2 g, resin)

As the complex formation is strongly pH dependent, careful adjustment of proper pH for the reagent was necessary. All the metal ions studied exhibited higher sorption capacity at pH 2.0-4.0±0.01 except for Co(II) and Zn(II) which show high sorption capacity at pH 4.0- 6.0±0.01. Hence, pH 4.0±0.01 was adjusted in all further experiments for Cu(II), Cr(III), Ni(II), Pb(II) except Co(II) and Zn(II) which were adjusted at pH 6.0±0.01.

119

thermostat shaker for pre-selected intervals of time. The loading halftime, t1/2, that is, the time required to reach 50% of the resins total loading capacity was evaluated from the resulting isotherm. From the kinetics of sorption for each metal (Figure 4.5), it was observed that 40 min was enough for the sorbent to reach the saturation level for all the metals. The sorption rate constant k can be calculated using the following equation: [20], -ln (1-F) = kt,

where F = Qt/Q and Qt is the sorption amount at sorption time t and Q the sorption

amount at equilibrium. Putting the value of Qt at t1/2 in the above equation we may get the corresponding value of k for every metal ion (Table 4.1).

Figure 4.5 Kinetics of sorption of metal ions on AXAD-4- HBAM (Experimental conditions: 50 mL, solution; 100 µg mL-1, metal ion; 0.2 g, resin) 120

Table 4.1 Kinetics and batch capacity of sorption of metal ions on AXAD-4-HBAM (experimental conditions: 50 mL, solution; 0.2 g, resin).

Loading halftime Rate constant Batch capacity Metal ion -1 -2 -1 t1/2 (min) k (min. ) ×10 (mmol g ) Cu(II) 5.5 12.54 0.27 Cr(II) 7.0 9.85 0.21 Ni(II) 8.0 8.62 0.18 Co(II) 9.0 7.66 0.15 Zn(II) 12.0 5.75 0.12 Pb(II) 15.0 4.60 0.10

Effect of resin amount on the sorption capacity To investigate the effect of the amount of the resin on the sorption of metal ions, an excess of the metal ion solution corresponding to 100 µg mL-1 was equilibrated with varying amounts of resin buffered at the suitable pH. The retention of the metal ions per gram of the resin increased with the increase in the amount of the resin. An almost constant and maximum sorption capacity was observed after 0.2 g of the resin.

Effect of flow rate for sorption and elution The flow rate for the sorption of metal ions was varied within the range of (1 to 10) mL min−1, and the results showed that there was no effect on the sorption of metal ions up to 5 mL min−1. However, at flow rates higher than 5 mL min−1, the sorption of metal ions decreased gradually. Hence, flow rate of 5 mL min−1 has been maintained for all further sorption studies. The stripping of the sorbed metal ions was found to be quantitative for eluent flow rate up to 3 mL min−1. Therefore, in the present work, the eluent flow rate of 3 mL min−1 has been maintained for the elution of metal ions. Such high flow rates for sorption as well as desorption support the superiority of the present resin over previously reported works [1,17,19].

Types of eluting agents In order to investigate the most efficient eluting agent, varying concentration of different volumes of HNO3, HCl and H2SO4 were tried. Other acids such as formic acid, acetic acid and perchloric acid were also investigated. Distilled water was found to be 121

unsuitable for the purpose of elution as <0.5% recovery was achieved indicating that the metal ions were retained by the resin by some strong bonding forces. For all the metal ions, 5 mL of 2 mol L-1 HCl was found to be satisfactory for quantitative recovery (>98%). Thus, in subsequent experiments, 5 mL of 2 mol L-1 HCl solution was used as eluent. Table 4.2 depicts the results. Such low concentration of the acid (eluting agent) prevents leaching of the functional groups, whereby enhancing the regenerability of the present sorbent.

Table 4.2 Percent recovery of metal ions from the AXAD-4-HBAM resin using different volume of varying concentration (Experimental condition: 3 mL min-1, elution flow rate) Stripping agent Mean recovery for five replicates (%) Concentration Volume Type Cu(II) Cr(III) Ni(II) Co(II) Zn(II) Pb(II) mol L-1 (mL) 1 5 52 48 55 60 50 42 HCl 2 5 100 99 100 99 98 99 1 5 40 35 45 38 42 51 H SO 2 4 2 5 55 46 58 50 52 58 1 5 56 50 45 42 51 38

HNO3 2 5 83 70 62 65 80 61 2 5 35 25 35 32 38 34 4 10 57 52 40 50 54 49 HCOOH 2 5 24 15 30 21 22 25 4 10 32 35 38 29 34 30 CH COOH 3 1 5 38 38 20 16 28 41 2 10 46 39 30 23 33 48 HClO 4 1 5 52 48 55 60 50 42

Resin reusability test For the reusability of the resin, the loading solution of the metal ions, having a concentration of 50 µg mL-1, was taken several times into a column while maintaining a flow rate of 5 mL min-1 and the sorbed metal ions were subsequently eluted by the appropriate eluting agent. The capacity of the modified resin was found to be apparently constant (less than 3%) after the repeated use of more than 40 cycles of sorption and desorption.

Study of interferences Various cations and anions, which are inevitably associated with heavy metals, may interfere in determination of the latter through precipitate formation, redox reactions, or

122

competing complexation reactions. In order to assess the analytical applicability of the resin to real samples, common chemical species such as sodium citrate, sodium tartrate , sodium - 2- + 2- 3- – + + oxalate, humic acid, fulvic acid, NO3 , CO3 , NH4 , SO4 , PO4 , Cl , K and Na were checked for any interference in the sorption of these metals. The effect of humic substances on metal-collection was examined because both humic and fulvic acids are generally present in natural waters at µg mL-1 to ng mL-1 levels and form complexes with various heavy metals [21-23]. Very few literatures [24,25] have considered the interference of these humic substances on the preconcentration of trace metal ions from natural waters. The tolerance limit is defined as the ion concentration causing a relative error smaller than ±5% related to the preconcentration and determination of the analytes. Many anions and cations, which are inevitably associated with metal ions present at the trace level in all natural waters, produce no interference in the sorption of the heavy metals up to appreciable concentrations (Table 4.3). A relative error of less than 5% was considered to be within the range of experimental error.

Table 4.3 Tolerance limit of foreign species (in binary mixtures) on sorption of metal ions (Experimental conditions: 50 mL, solution; 10 µg, metal ion; 0.2 g, resin) Foreign species Tolerance limit of metal ions (µg mL-1) Cu(II) Cr(III) Ni(II) Co(II) Zn(II) Pb(II) NaCl 35000 30000 35000 35000 35000 30000

Na2SO4 5000 5000 5000 5000 5000 4000

NaNO3 20000 20000 20000 20000 20000 18000

Na3PO4 1200 1000 1200 1200 900 600

NH4Cl 35000 35000 35000 35000 30000 25000 Sodium citrate 250 200 200 200 250 150 Sodium oxalate 100 85 100 100 100 80 Sodium potassium tartrate 65 100 150 120 90 65

CH3COONa 15000 15000 15000 15000 15000 12000

CaCl2 35000 30000 35000 35000 32000 25000

MgCl2 32000 30000 32000 32000 32000 28000 Humic acid 35 55 50 50 40 55 Fulvic acid 30 45 45 40 35 45

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Figure 4.6 Langmuir sorption isotherms depicting the sorption behaviors of metal ions onto AXAD-4-HBAM (Column parameters: 5 mL min-1, sorption flow rate; 0.2 g, resin)

Adsorption isotherm In order to determine the sorption capacity of the resin, test solutions of Cu(II), Cr(III), Ni(II), Co(II), Zn(II) and Pb(II) weighing in the range (100 to 50,000) µg were taken in batches containing 0.2 g of chelated resin and recoveries were investigated. Langmuir isotherms were plotted in order to determine the resin capacity in accordance to the literature [26]. The Langmuir adsorption isotherm is described by the equation given as,

qe=qmaxaLCe/(1+ aLCe)

−1 where qe is the amount of metal sorbed per unit weight of the resin (mg g ) at

−1 equilibrium, Ce is the final concentration in the solution (mg L ), qmax is the maximum

−1 sorption at monolayer coverage (mg g ), and aL is the sorption equilibrium constant, which is related to the energy of sorption. A plot of Ce/qe versus Ce shows linearity and hence, the

Langmuir constants, qmax and aL, can be calculated from the slope and intercept of the plot.

From the plots obtained for each metal, the amount of maximum total metal (qmax) sorbed on 1.0 g of resin is calculated. The maximum sorption capacity calculated from the Langmuir 124

isotherm (Figure 4.6) indicated that the resin retained Cu(II) more strongly than the other metal ions under study. As compared to other works [Table 9], the sorption capacity of this modified resin is higher. The higher surface area of AXAD-4 resin complemented by highly efficient incorporation of HBAM may be the contributing factors for higher sorption. The other most probable factor may be the high hydrophilicity of AXAD-4-HBAM which facilitates better surface contacts with the metal ions in aqueous solutions.

The Freundlich isotherm postulates heterogeneous surface energy system and hence is the most convenient form of representing the experimental data obtained for different particle sizes [27]. Therefore, the Freundlich isotherm was also modeled to fit the equilibrium data and is given by the following equation

ln qe = ln KF + ln Ce

where KF and 1/n are Freundlich constants related to adsorption capacity and energy of adsorption, respectively. However, as depicted in Figure 4.7, the low R2 value, obtained for each metal, suggests that the data do not fit well onto Freundlich adsorbtion isotherm.

Figure 4.7 Freundlich sorption isotherms depicting the sorption behaviors of metal ions onto AXAD-4-HBAM (Column parameters: 5 mL min-1, sorption flow rate; 0.2 g, resin)

125

Preconcentration factor and breakthrough capacity The limit of preconcentration was determined by increasing the volume of metal ion solution and keeping the total amount of loaded metal ion constant at 10 µg. The breakthrough volume corresponds to the volume at which the effluent concentration of metal ions from the column is about (3 to 5) % of the influent concentration. The breakthrough volume was determined by the dynamic procedure. The overall capacity, breakthrough capacity and the degree of utilization was determined by the literature method [28]. Figure 4.8 gives the breakthrough curves for all the metal ions studied. The overall sorption capacity calculated on the basis of total saturation volume was compared with the corresponding breakthrough capacities for each metal.

Figure 4.8 Breakthrough curves for sorption of metal ions: C/Co is the concentration ratio of the effluent to influent (Column parameters: 5 mL min-1, sorption flow rate; 0.5 g, resin)

The closeness of the dynamic capacity to the total sorption capacity reflects the applicability of the column technique for preconcentration. Quantitative collection of metal ions was possible from solutions of concentration in the order of 5.0-6.3 ng mL-1 with a

126

recovery up to 98% resulting in a preconcentration factor of 320-400 (Table 4.4) which is much higher than many previous works [Table 4.9].

Table 4.4 Preconcentration and breakthrough profiles of metal ions on AXAD-4-HBAM (Experimental conditions: 5 mL min-1, sorption flow rate; 3 mL min-1, elution flow rate; 0.5 g, resin) Preconcentration studies Breakthrough studies Degree Concentra Overall Breakthrou Breakthro Metal Total Precon- of tion sorption gh ugh ions volume centration column limit capacity capacity volume (mL) factor utilizati (ng mL-1) (mmol g-1) (mmol g-1) (mL) on Cu(II 2000 5.0 400 0.29 0.24 1700 0.82 Cr(II) 1900 5.3 380 0.1 8 1600 0.82 0.22 Ni(II) 1900 5.3 380 0.20 0.16 1600 0.80 Co(II 1800 5.6 360 0.16 0.12 1500 0.75 Zn(II 1600 6.3 320 0.09 1400 0.72 0.13 Pb(II) 1600 6.3 320 0.08 1400 0.72 0.11

4.4 Method validation

Prior to analysis of real water samples, validation of the method was performed analyzing standard reference materials and recoveries of trace metals after spiking. In order to test the accuracy of the method, 50 mL of pretreated environmental (vehicle exhaust particulates), biological (human hair, Chlorella and tea leaves) and alloys (rompin hematite and zinc base die-casting alloy C) standard reference material samples were analyzed by recommended column method after adjusting its suitable pH. The mean concentration values of the metals studied agreed with the certified values. Calculated Student’s t (t-test) values for respective metal ions were found to be less than critical Student’s t-values at the 95% confidence level (Table 4.5). Hence, the mean values were not statistically significant from the certified values indicating absence of bias in the present method. Analytical recoveries of metal ions were ascertained by measuring the recovery of the spiked quantity (5 µg) from 100 mL of synthetic mixture containing 5 µg of each of the other metal ions studied using recommended column method with 0.2 g of the chelating resin. 127

These concentrations were guided by middle value of preconcentration limit and maximum concentration of working range of calibration curve of FAAS for each metal ion in order to ensure complete sorption and avoid dilution of the final eluate during determination. It was found that the mean percentage recoveries of all the metal ions studied were (99.5 to 101.5) % with relative standard deviation (RSD) <5%. Precision of the proposed method is reflected by low RSD (<5%) in the analysis of SRMs as well as various water samples (Table 4.5 and 4.6).

Table 4.5 Analysis of metal ions in standard reference materials (Experimental conditions: 50 mL, solution; 5 mL min-1, sorption flow rate; 3 mL min-1, elution flow rate; 0.2 g, resin) Calculated Certified value Found by proposed method Samples Student's ‘t’ (µg g-1) µg g-1 (RSD )a value b Co: 3.2±0.25, Cu: 65.7±1.72 Vehicle exhaust Co: 3.3, Cu: 67, Ni: 1.93,1.44, Ni: 18.2±0.38, Pb: particulates 18.5, Pb: 219.0, Zn: 1.18, 1.50, 217.7±3.41 NIES 8 c 1040 1.21 Zn: 1035.8±10.72 Zn: 167.3±2.21, Cu: Human hair Zn: 169.0, Cu: 16.3, 1.58,1.45, 15.8±0.80, Ni: 1.7±0.11, Pb: NIES 5 Ni: 1.8, Pb: 6.0 1.34, 1.72 5.7±0.28 Chlorella Zn: 20.5, Cu: 3.5, Co: Zn: 19.7±0.77, Cu: 3.3±0.18, 1.67, 1.77, NIES 3 0.87, Pb: 0.60 Co: 0.9± 0.03, Pb: 0.6±0.05 1.81, 1.55 Tea leaves Zn:33.0, Zn:32.0±1.03, 1.98, NIES 7 Cu: 7.0, Ni:6.5 Cu: 6.9±0.22, Ni:6.4±0.14 1.79,1.28 Rompin Cu: 633.7±9.21, Cu: 640.0, Zn: 1030.0, 1.89, hematite, Zn: 1021.2±14.34 Pb: 210.0 2.01, 1.32 JSS (800-3) d Pb: 206.9±3.86 Zinc base die- Cu: 1320.0, Cu: 1312.6±11.10, 1.88,1.57, casting alloy C NBS Pb:82.0,Ni: 29.0 Pb:80.2±1.56, Ni: 27.7±1.35 1.30 627 e a x ± standard deviation, n = 5 ; b at 95 % confidence level; c National Institute of Environmental studies (NIES); d Iron and steel institute of Japan (JSS); e National bureau of Standards (NBS)

128

The calibration curves were found to be linear over the concentration ranges 0.0058- 0.500, 0.0062-0.7500, 0.0062-1.000, 0.0062-0.750, 0.0066-0.010, and 0.0071-1.500 µg mL-1 for Cu(II), Cr(III), Ni(II), Co(II), Zn(II) and Pb(II), respectively when the standard solutions were prepared in 100 mL. The regression equations and correlation coefficients (R2), obtained by the method of least squares, were A=0.0629C + 0.0033 (R2= 0.9999), A=0.0222C + 0.0043 (R2= 0.9997), A=0.0608C + 0.0019 (R2= 1), A=0.0448C + 0.0020 (R2= 0.9999), A=0.0971C + 0.0020 (R2= 0.9997) and A=0.0372C + 0.0028 (R2= 0.9997) for Cu(II), Cr(III), Ni(II), Co(II), Zn(II) and Pb(II), respectively, where A is the absorbance and C is the metal ion concentration (µg mL-1). The linearity of the calibration curves is apparent from the correlation coefficients (R2) which lie well above 0.999.

Blank runs were performed for 20 times by applying the recommended column procedure with 100 mL of aqueous solution prepared by adding suitable buffer (excluding metal ions) and finally eluting the same in 5 mL before subjecting it to FAAS determination. The detection limits, evaluated as three times the standard deviation (s) of the blank signal, were found to be 0.39, 0.49, 0.42, 0.59, 0.71, and 1.10 ng mL-1 for Cu(II), Cr(III), Ni(II), Co(II), Zn(II) and Pb(II), respectively.

4.5 Applications

Applicability of the present method for preconcentration and determination of metal ions was accomplished by analyzing 500 mL of each river, canal, sewage and tap water after adjusting to suitable pH. The concentrations of metal ions were determined by following recommended column method using FAAS (direct method). The metal determinations were also confirmed using the standard addition method (S.A) in which 1 µg of each metal ion is added to 500 mL of each water sample and adjusted to suitable pH. The concentrations of metal ions were then determined by following recommended column method using FAAS (S.A. method). The closeness of results of direct and S.A. method (Table 4.6) indicates the reliability of the present method for metal analysis in water samples of various matrices.

129

Table 4.6 Determination of metal ions in natural waters collected from various locations after preconcentration by AXAD-4-HBAM column (Experimental conditions: 500 mL, solution; 5 mL min-1, sorption flow rate; 3 mL min-1, elution flow rate; 0.5 g, resin) Metal ion found by proposed method (µg L-1)± Standard deviationb Samples Method (%recovery of the spiked amount) Cu(II) Cr(III) Ni(II) Zn(II) Co(II) Pb(II) Canal Direct 10.2±0.18 5.7± 0.34 6.7±0.12 5.9±0.28 5.3±0.15 - water S.Ac 10.3±0.24 6.1±0.39 6.6±0.10 6.0±0.35 5.3±0.16 1.9±0.11

Tap Direct 8.5±0.20 9.7±0.29 4.7±0.15 8.8±0.24 5.8±0.20 6.8±0.23 water S.A. 8.5±0.25 10.0±0.33 4.7±0.18 8.9±0.27 5.8±0.27 6.8±0.25

Sewage Direct 6.8±0.29 7.5±0.21 13.2±0.31 6.7±0.20 6.2±0.16 4.8±0.19 water S.A. 6.9±0.32 7.8±0.28 13.3±0.38 6.7±0.30 6.4±0.18 5.0±0.20

River Direct 12.4±0.25 8.1±0.26 5.7±0.23 9.8±0.35 7.2±0.25 - water S.A. 12.6±0.22 8.2±0.19 5.7±0.25 10.1±0.33 7.3±0.08 2.2±0.10 a recommended procedure applied without spiking; b Average of five determinations; c recommended procedure after spiking (standard addition method); ‘-‘ indicates not detection

Table 4.7 Determination of metal ions in multi-vitamin capsule, infant milk substitute and hydrogenated oil (experimental conditions: 50 mL, solution; 5 mL min-1, sorption flow rate; 0.2 g, resin) Reported value Found by proposed method (µg g-1 )±standard Samples (µg g-1) deviation a Maxirich (Cipla) Cu: 398.2; Zn: Cu: 393.6±10.21, Zn: 439.2±11.15 Lactogen Cu: 2.9; Zn: 37.0 Cu: 2.82±0.09, Zn: 35.4±1.54 1(Nestle) Vanaspati ghee Ni: 0.45 Ni: 0.41±0.007 a Average of five determinations

The proposed method has also been applied for the determination of metal ions in multivitamin formulation, IMS vanaspati ghee (Table 4.7), fish and urine (Table 4.8). The observed results obtained were found to be accurate and in close agreement with the reported value (Table 4.7). 130

Table 4.8 Analysis of Common carbs (Cyprinus carpio) and urine for metal content (experimental conditions: 50 mL, solution; 5 mL min-1, sorption flow rate; 0.2 g, resin) Metal ions Fish Urine Muscles Livers Gills Found Recovery (µg g-1) a (µg g-1) a (µg g-1) a (µg L-1)a (%)

Cu(II) 0.112±0.002 0.263±0.003 0.523±0.005 15.64±1.34 101.4

Zn(II) 1.211± 0.07 2.168± 0.19 2.784± 0.38 127.38±1.22 99.0 Pb(II) 0.188± 0.002 0.245±0.002 0.338±0.003 12.75±1.06 100.0 Ni(II) 0.124±0.002 0.102±0.002 0.179±0.002 4.95±0.92 98.5 a (X ± S.D)

4.6 Conclusion The chelating ability of HBAM has been utilized in developing chelating sorbents for the purpose of separation and preconcentration of trace metal ions. The results reflect its promising nature for trace metal ion analysis in various natural water resources, environmental and biological samples. The main advantages of this procedure are the simple and fast preparation of the chelating resin and no requirement of organic solvents in the metal elution step. The excellent ability for the exclusion of alkali and alkaline earth elements on the AXAD-4-HBAM resin makes it desirable for use in the separation and preconcentration of trace elements because their presence often interfere in the subsequent FAAS determination. The results obtained demonstrated good reproducibility. Overall, AXAD-4- HBAM has a higher sorption capacity, preconcentration factor, preconcentration limit, detection limit and tolerance limits which are superior in comparison to previous works (Table 4.9) except one or two metal ions have high sorption capacity [29] and preconcentration limit [30]. Moreover, the use of a column preconcentration technique allows for the assessment of low trace metal concentrations, even by less sensitive determination methods such as FAAS. Preconcentration by this material (AXAD-4-HBAM) from river water samples do not require any prior digestion of the samples. It can be successfully applied for the analysis of both environmental and biological samples as indicated by the high precision (low relative standard deviation).

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Table 4.9 Comparison of previous published works with present work using various chelating resins in terms of different parameter Sorption Detection Type of support Metal ions capacity Preconcent Preconcentration limit (mmol g-1) ration factor Limit (ng mL-1) (ng mL-1) References Cu(II), Cr(II), 0.27, 0.21, 400, 380, 0.39, 0.49, Amberlite Ni(II), 5.0, 5.3, 5.3, 5.6, Present 0.18, 0.15, 380, 360, 0.42, 0.59, XAD-4-HBAM Co(II), 6.3, 6.3 work 0.12, 0.10 320, 320 0.71, 1.10 Zn(II), Pb(II) Silica- Cu(II) 1.12 - - 6.0 1 Dimethylglyoxime Cd(II), 0.007, XAD-4-aluminon - - 0.02, 0.11 17 Zn(II) 0.014 Amberlite Co(II) - 200 - 0.24 19 XAD-7- BPMBDA Cd(II), 0.197, Amberlite Co(II), 0.107, 200, 180, 0.48, 0.20, 20, 5, 20, 2.5, 5, XAD-2-Thiosalicylic Cu(II), 0.214, 200, 400, 4.05, 0.98, 20 29 acid Fe(II), 0.066, 200, 200 1.28, 3.94

Ni(II), 0.309, Zn(II) 0.047 Pb(II), 0.059, Amberlite Cd(II), 0.08, 400, 400, XAD-4-o- 2.5, 2.5, 6.5, Co(II), 0.091, 150, 200, - 30 aminobenzoic acid 5.0, 2.5 Ni(II), 0.121, 400

Zn(II) 0.116 Cu(II), 0.053, Amberlite Co(II), 0.058, 50, 50, 4.0, 2.0, XAD-2-o- Cd(II) 0.028, 20, 10, 10, 20, 100, 65, 5.0, 7.5, 31 aminophenol Ni(II), 0.055, 10, 25 40, 40 2.5, 25 Zn(II), 0.044, Pb(II) 0.016 Amberlite Zn(II), 0.018, 140, 180 5.5, 7.0 - 32

132

XAD-2-salicylic Pb(II) 0.002 acid Cd(II), 0.041, Amberlite Co(II), 0.023, 200, 200, XAD-2- Cu(II), 0.093, 5, 10, 20, 25, 100, 80, - 33 pyrocatechol Fe(II), 0.074, 10, 10 200, 200 Ni(II), 0.053, Zn(II) 0.028 Pb(II), Amberlite Ni(II), 100, 150, - 1.6, 0.2, XAD-1180- - 34 Cu(II), 150, 150 0.3, 0.3 salicylaldoxime Mn(II) 0.06, Co(II), 0.08, Cu(II), - - Silica- 0.05, 40, 40, Fe(II), 35 salicylaldoxime 0.04, 40, 40, 40 Ni(II), 0.04 Zn(II)

Cellulose acetate- 8- Ni(II) - 20 - 4.87 36 hydroxyquinoline

133

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Chapter 5

A newly developed salicylanilide functionalized Amberlite XAD-16 chelating resin for its use in preconcentration and determination of trace metal ions from environmental and biological samples

5.1 Introduction

The synthesis of chelating resins has been attracting considerable interest for the preconcentration and separation of trace elements from various real samples of environmental and biological origin prior to their determination [1-11]. Flame atomic absorption spectrophotometry coupled to preconcentration procedures is an advantageous technique because of its simple detection system based on flame atomization instead of a flameless technique, which require more expensive equipment and are usually much more sensitive to interferences from matrix [12-22]. The need for preconcentration arises due to the very low concentration of metal ions as well as matrix effect that poses serious interferences in the determination of metal ions even by sophisticated analytical instruments [17,23-28]. The selective removal of toxic metal ions and recovery of precious metal ions in terms of environmental protection and economic consideration are of great significance [6, 26,29-30]. Chelating resins suited to perform this task should possess significant selectivity, enhanced hydrophilicity [31], better metal loading capacity and regenerability. Their selectivity is affected by chelation ability of immobilized multidentate ligands, nature and degree of cross-linking of a polymeric support [13,15,16,32-36], the pH of the solution and the kinds of the metal ions. The hydrophobicity of these chelating resins may be overcome by introducing hydrophilic functional groups thereby enhancing its surface contact with aqueous phase metal ions which facilitates efficient column operation [11,37,38]. Capacity may be improved by increasing the number of chelating sites as well as the accessibility of metal ions to these sites. These twin objectives can be achieved by selecting a ligand containing many non-sterically hindered chelating moieties, and a polymeric support of high surface area and good porosity. Amberlite XAD 16 resins would serve this purpose as the copolymer backbone for the immobilization of chelating ligands because of its high surface area compared to other Amberlite XAD series resins in addition to inherent superior physical properties such as porosity, uniform pore size distribution and chemical stability [37-39]. In the present study, a new chelating resin (AXAD-16-SALD) has been synthesized chemically by functionalizing Amberlite XAD-16 resin with salicylanilide (Figure 5.1) through an azo spacer arm with an aim to find an efficient sorbent for the separation and preconcentration of metal ions from environmental and biological samples before its

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subsequent determination by FAAS. The main objective for choosing this ligand is its capability of forming chelates with metal ions through its three hydrophilic binding sites (- OH, >C=O, -N-H) [40]. Hence, the method is validated by analyzing standard reference materials (SRMs) and carrying out recovery experiments before applying it to the analysis of natural water, mango pulp, leafy vegetables and fishes.

(CH CH2)n

b N Mn+

N a Mn+ OH

H Figure 5.1 Structure of a monomeric unit of AXAD-16 modified with Salicylanilide; (a) and (b) are the probable chelating sites

5.2 Experimental Both batch and column methods (Section 2.6) have been employed for the sorption and elution studies of Cu(II), Co(II), Ni(II), Zn(II), Cr(III), Cd(II), and Pb(II) prior to their determination by flame atomic absorption spectrometry (FAAS). The recommended procedures (Section 2.5-2.6) have been applied for the determination of different experimental parameters, including physico-chemical properties, of the resin.

The physico-chemical properties that have been determined include:

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The experimental parameters that have been optimized include:

 Optimum pH range for sorption was ascertained by applying the recommended procedure (Section 2.6.1)  Contact /half-loading time for sorption has been determined (Section 2.6.1)  Flow rate for sorption as well as for elution has also been determined (Section 2.6.3)  Eluting agent for complete desorption (Section 2.6.3)  The recommended procedure (Section 2.6.2) was followed to determine the preconcentration factor  Breakthrough volume for sorption has been determined according to the recommended procedure (Section 2.6.2)  The recommended procedure (Section 2.6.2) was followed for the determination of limit of detection (LOD) for each metal.

The following experiments have been performed for the validation of the method:

The method has been applied for the following applications:

 Collection and pretreatment (Section 2.3.1) of natural waters prior to determination of trace metal ions  Determination of Cu(II) and Zn(II) in multivitamin formulation and infant milk substitute was made after digestion of the samples according to the recommended procedure as in section 2.3.2.

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 Digestion of hydrogenated oil was carried out (Section 2.3.2) prior to the determination of Ni(II) content.  Digestion of standard reference materials were carried out according to the recommended procedure as in section 2.3.3.  The recommended procedure (Section 2.3.4) was followed for the digestion of fish samples prior to their analysis for metal content.  The recommended procedure (Section 2.3.5) was followed for the digestion of mango pulp and mint leaves prior to their analysis for metal content.

5.3 Results and discussion

5.3.1 Characterization of the chelating resin The extent of the coupling reaction may be interpreted by subjecting the products, formed at each step, to elemental analysis. The nitrogen content of the nitrated resin and the subsequent reduced product (aminated resin) was found to be 9.12% and 11.28%, respectively. Elemental analysis of AXAD-16-SALD gave 68.25%, 5.46%, and 11.02% of C, H and N, respectively, which agrees well with the calculated values, considering the possible stoichiometry of its repeat unit to be C21H18N3O2.H2O (C, 69.61%, H, 5.52% and N, 11.60%). The presence of 11.02% of N suggests the incorporation of at least 2.65 mmol of salicylanilide if we assume that all the functional groups have been attached through azotisation. In thermogravimetric analysis (Figure 5.2), the AXAD-16-SALD resin shows an earlier weight loss of 5.2 % up to 180 °C. This initial step corresponds to the endothermic peak in the DTA curve which may be attributed to the loss of coordinated water molecule. The TGA and elemental analysis together suggest that at least one water molecules per repeat unit is sorbed. The subsequent exothermic peak corresponding to the weight loss of 12.82% up to 215 °C may be due to the loss of functional groups. Thermal analysis indicated that the synthesized resin was stable up to 200 °C, above which degradation commences. The sorption capacity observed for the resin, which had been subjected to heating upto 215 °C before cooling to the required temperature, was found to reduce by 23%.

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Figure 5.2 TGA/DTA curves of AXAD-16-SALD

The IR spectrum of the modified resin AXAD-16-SALD (Figure 5.3) was compared with that of untreated Amberlite XAD-16. Besides the band (3479 cm-1) for -OH group, there are three additional bands at 1695, 1649 and 1448 cm-1 which may be assigned to carbonyl (>C=O), azo (–N=N–) stretching vibrations and (-N-H bending), respectively. On comparing the IR spectrum of the metal ion-free chelating resin with that of the metal ion-saturated resin, a red shift (5–10cm-1) in the bands corresponding to -OH group and ketonic group of salicylanilide is exhibited in the latter. This suggests that chelation of metal ions through – OH and ketonic groups of reagents (SALD) are probably responsible for metal sorption. The availability of two chelation sites on the resin probably results in its high metal ion sorption capacity. The IR spectrum of the resin, which had been stripped off the metal ions, shows resemblance with the IR spectrum of metal free resin. The resin showed no loss in sorption capacity up to the concentration of 5 mol L-1 of -1 acids, such as HCl, HNO3 and H2SO4, and alkaline medium constituting 5 mol L NaOH. Hence, the resin exhibited high chemical stability. 141

Figure 5.3 FT-IR spectrum of a) AXAD-16-SALD and b) AXAD-16-SALD saturated with Cu(II)

The water regain capacity was found to be 12.90 mmol g-1. This value reflects the high hydrophilicity of the resin which is satisfactory for column operation. The overall hydrogen ion capacity amounts to 6.08 mmol g-1 of resin, which may be contributed both by the hydroxylic and amide groups present within the molecule. Theoretically, if 2.65 mmol of the reagent constituted per gram of the resin, the hydrogen ion capacity, due to the hydroxylic group should have been 2.65 mmol g-1, which agrees well with the experimental -1 value (2.53 mmol g ) corresponding to hydrogen ion capacity with NaHCO3.

5.3.2 Optimum experimental parameters In order to optimize sorption of metal ions, the univariate approach was followed to establish all the parameters. Each optimum condition was established by varying one of them and following the recommended procedure.

Preliminary experiments showed that the maximum sorption of Cu(II), Co(II), Ni(II), Zn(II), Cr(III), Cd(II), and Pb(II) (Figure 5.4) was observed at pH 9.0, 9.0, 9.0, 9.0, 9.0, 6.0, and 6.0, respectively. Hence, pH 9.0 for Cu(II), Co(II), Ni(II), Zn(II), Cr(III) and 6.0 for Cd(II), and Pb(II) were adjusted in all further experiments.

Figure 5.4 Dependence of sorption capacity on the pH of the solution (Batch method parameter: 100 mL, solution; 100 µg mL-1, metal ions; 0.2 g, resin)

Effect of buffer volume on sorption For the sorption of metal ions the test solution was buffered with appropriate volume of the buffer solution for maintaining the pH. The different volume 0.5-10 mL of buffer solution was added in the test solution and it was observed that 5 mL of buffer solution is sufficient to reach the saturation level. Hence, a 5 mL of buffer solution was added for all subsequent experiments.

Kinetics of sorption To study the effect of time on the sorption, resin was stirred with solution containing one of the metal ions (20 µg mL) at different temperatures for 2, 5, 10, 15, 20, 30, 60, 90 and

120 min (under the optimum conditions). The loading half time (t1/2) needed to reach 50% of the total loading capacity was estimated from Figure 5.5 and reported in Table 5.1. From the kinetics of sorption for each metal (Figure 5.5), it was observed that 20-30 min was enough

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for the sorbent to reach the saturation level for all the metals which reflects good accessibility of the chelating sites in the resin.

Figure 5.5 Kinetics of sorption of metal ions on AXAD-4-SALD (Batch method parameter: 100 mL, solution; 100 µg mL-1, metal ions; 0.2 g, resin)

Table 5.1 Kinetics and batch capacity of sorption of metal ions on AXAD-16-SALD (experimental conditions:100 mL, solution; 0.2 g, resin) Batch Loading Rate Metal constant -ΔH/ ΔS/ capacity/ halftime -2 -1 -1 -1 ions -1 a k x10 /min J mol ± S.D J mol K ± S.D µmol g ± S.D t1/2/min -1

Cu(II) 770.2±0.35 3.5 19.7 67.85±0.52 117.00±0.97 Co(II) 732.5±0.36 4.2 16.4 45.65±0.48 82.46±0.58 Ni(II) 718.2±0.29 5.0 13.8 42.18±0.41 77.01±0.44 Zn(II) 631.8±0.31 6.3 10.9 55.75±0.51 96.03±0.65 Cr(III) 613.2±0.38 7.5 9.2 52.18±0.46 89.59±0.55 Cd(II) 290.3±0.18 9.5 7.8 57.96±0.62 97.33±0.79 Pb(II) 151.8±0.26 11.0 6.8 58.47±0.68 101.68±0.88

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The kinetic studies also showed that the temperature affected the rate constants significantly; that is, saturation was reached at a faster rate at higher temperature. This temperature effect may be a manifestation of the fact that the resin swells more completely at higher temperature, which allows metal ions to diffuse more easily into the interior of the resin, and that the sorption was an endothermic process and hence high temperature facilitates higher sorption. A plot (Figure 5.6) of log D versus 1/T, where the distribution ratio (D) represents the ratio of sorption capacity and the concentration of free metal ion at the equilibrium sorption, respectively, revealed that the distribution ratio increased with the increase of temperature. This again implies that the sorption process was an endothermic process. The values of ΔH and ΔS were calculated (Table 5.1) using the slope and intercept from the above plots using the following relationship [36]:

− ∆H ∆S log D = − + (1) 2.303RT 2.303R

Figure 5.6 Influence of temperature on the distribution ratio of metal ions (Batch method parameter: 100 mL, solution; 20 µg mL-1, metal ions; 0.2 g, resin)

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The dynamics of the adsorption process in terms of the order and the rate constant can be evaluated using the kinetic adsorption data. The process of the mention metal ions removal from an aqueous phase by any adsorbent can be explained by using kinetic models and examining the rate-controlling mechanism of the adsorption process such as chemical reaction, diffusion control and mass transfer. The kinetic parameters are useful in predicting the adsorption rate which can be used as important information in designing and modeling of the adsorption operation. The kinetics of removal of such ions is explicitly explained in the literature using pseudo first-order, second-order kinetic models [41-42]. k t Pseudo-first-order model: log(q − q ) =log q − 1 (2) e t e 2.303 t 1 t = + Pseudo-second-order model: 2 (3) qt k2 q e qe

Where k1 and k2 is the rate constant of pseudo first-order and pseudo-second- order

rate constant, respectively. qe and qt are adsorption capacity at equilibrium (mmol/g) and at anytime, respectively. The parameters obtained from the kinetics models are listed in Table 5.2.

Table 5.2 Kinetics parameter for sorption of different metal ions (experimental conditions:100 mL, solution; 0.2 g, resin) Order of Parameters Cu(II) Co(II) Ni(II) Zn(II) Cr(III) Cd(II) Pb(II) reactions -1 K1 (min ) 0.269 0.185 0.153 0.142 0.137 0.130 0.122 Pseudo-first- -1 qe (mmol g ) 0.592 0.504 0.520 0.446 0.312 0.181 0.102 order model R2 0.9742 0.9596 0.9715 0.9467 0.9688 0.9571 0.9268

K2 Pseudo- 32.25 27.47 15.15 13.13 10.97 9.14 7.72 (g/mmol.min) second-order -1 qe (mmol g ) 0.760 0.725 0.720 0.623 0.612 0.288 0.150 model R2 0.9998 0.9995 0.9995 0.9994 0.9996 0.9994 0.9991

It is evident from Table 5.2 and Figure 5.7a and b that a more precise fit of kinetics

data was shown by the pseudo-second order model. The calculated qe values are closer to the 146

experimental data than the calculated values of pseudo-first-order model and the values of regression coefficients (R2) are higher (0.99) than pseudo-first-order kinetic model.

Figure 5.7a, b Pseudo first and second-order kinetic plots for removal of metal ions (Batch method parameter: 100 mL, solution; 20 µg mL-1, metal ions; 0.2 g, resin) 147

Adsorption isotherm For an adsorption column, the column resin is composed of microbeads. Each binding particle immobilized to the micro bead can be assumed to bind in a 1:1 ratio with the solute sample passed through the column. At the concentration range (1 x 103 – 1 x 104 µg) studied for Cu(II), Co(II), Ni(II), Zn(II), Cr(III), Cd(II), and Pb(II) the data were successfully applied for Langmuir isotherm. The Langmuir model assumes that sorption occurs on defined sites of the sorbent with no interaction between the sorbed species and that each site can accommodate only one molecule (monolayer adsorption) with the same enthalpy sorption, independent of surface coverage. The linearized form of Langmuir isotherm may be represented by the following equation [43].

Ce 1 Ce = + q q b q e m m (4)

Figure 5.8 Langmuir sorption isotherms depicting the sorption behaviors of metal ions onto AXAD-16-SALD. (Column parameters: 4 mL min-1, sorption flow rate; 0.2 g, resin)

-1 Here, Ce is the equilibrium concentration (mg L ), qe is the amount adsorbed at -1 equilibrium (mg g ) and qm and b is Langmuir constants related to adsorption efficiency and energy of adsorption, respectively. The linear isotherm occurs when the solute concentration 148

is very small relative to the binding molecule of the solid phase. The linear plots of Ce/qe

versus Ce suggest the applicability of the Langmuir isotherms (Figure 5.8).

The values of qm and b were determined from slope and intercepts of the plots and are

presented in Table 5.3. From the values of adsorption efficiency, qm we can conclude that the maximum adsorption corresponds to a saturated monolayer of adsorbate molecules on adsorbent surface with constant energy and no transmission of adsorbate in the plane of the adsorbent surface. To confirm the favorability of the adsorption process, the separation factor

(RL) is calculated as presented in Table 5.3. The values were found to be between 0 and 1 which confirm the favorability of the adsorption process.

Table 5.3 Langmuir isotherm constants for sorption of metal ions (Column parameters: 5.0 mL min-1, sorption flow rate; 0.2 g, resin) Standard Metal b qm 2 RL R deviation ions (L mg-1) (mg g-1) (N=5) Cu(II) 0.9876 0.0055 48.45 0.9958 0.3572 Co(II) 0.9398 0.00539 42.98 0.9976 0.4150 Ni(II) 0.8897 0.00515 42.15 0.9988 0.1936 Zn(II) 0.8599 0.00870 41.21 0.9992 0.3404 Cr(III) 0.8792 0.01533 31.62 0.9993 0.2648 Cd(II) 0.7945 0.01919 32.64 0.9997 0.5348 Pb(II) 0.9289 0.01522 31.65 0.9999 0.8733

mL min-1. The decrease in sorption, or exchange, with increasing flow rate is due to the decrease in equilibration time between two phases. In the elution studies, 100% recovery of the sorbed metals from the resin could be achieved up to a flow rate of 3.0 mL min-1and hence, maintained for further studies. . Type of eluting agents and resin reusability test Quantitative elution of metal ions was studied with different mineral acids namely -1 H2SO4, HCl and HNO3 using varying volumes (1-10 mL) and concentrations (1-5 mol L ).

Among the acids, H2SO4 and HNO3 could elute 90% and 95%, respectively, when a maximum of 10 mL (5.0 mol L-1) each were used. When 10 mL of 4 mol L-1 HCl was used, almost complete desorption (>99%) was observed. The efficacy of the eluent (4 mol L-1 HCl) was studied taking its different volumes (1-10 mL). It was found that 3-5 mL of acid was sufficient for quantitative elution (>99%) of all the metal ions. The resin was subjected to several loading and elution cycles by the dynamic method. -1 The resin bed can be regenerated fully with 4 mol L HCl easily and can be regenerated fully up to 55 cycles. Therefore, multiple use of the resin is feasible. Similar results are shown by batch method also.

Study of interferences Various cations and anions may interfere in the determination of metal ions through precipitate formation, redox reactions, or competing complexation reactions. In order to assess the analytical applicability of the resin, various common chemical species were checked for any interference. The effect of humic substances on metal-collection was also examined because both humic and fulvic acids are generally present in natural waters at µg mL-1 to ng mL-1 levels and form complexes with various heavy metals [44-46]. Very few literatures [47,48] have considered the interference of these humic substances on the preconcentration of trace metal ions from natural waters. The tolerance limit is defined as the ion concentration causing a relative error smaller than ± 5 % related to the preconcentration and determination of the analytes. Many anions and cations, which are inevitably associated with metal ions present at the trace level in all natural waters, produce no interference in the sorption of the heavy metals up to appreciable concentrations (Table 5.4).

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Table 5.4 Tolerance limit of foreign species (in binary mixtures) on sorption of metal ions (Column parameter: 100 mL, solution; 10 µg, metal ion; 0.2g, resin) Foreign species Tolerance ratio(Foreign species / Metal ion ) (µg mL-1) Cu(II) Co(II) Ni(II) Zn(II) Cr(III) Cd(II) Pb(II) Na+ (NaCl) 3.93 × 105 3.14 × 105 3.14 × 105 2.35 × 105 1.57 × 105 1.57 × 105 0.78 × 105 K+ (KCl) 5.24 × 105 4.19 × 105 3.14 × 105 4.19 × 105 2.09 × 105 2.09 × 105 1.04 × 105 + 5 5 5 5 5 5 5 NH 4 (NH4Cl) 2.97 × 10 2.38 × 10 2.38 × 10 2.97 × 10 1.78 × 10 1.19 × 10 2.37 × 10 2+ 4 4 4 4 4 4 4 Mg (MgCl2) 2.55 × 10 2.04 × 10 2.04 × 10 2.55 × 10 1.53 × 10 1.02 × 10 0.51 × 10 2+ 4 4 4 4 4 4 4 Ca (CaCl2) 3.61 × 10 2.88 × 10 3.61 × 10 2.16 × 10 1.44 × 10 1.44 × 10 0.72 × 10 Cl- (NaCl) 6.06 × 105 4.85 × 105 6.06 × 105 4.85 × 105 3.63 × 105 2.42 × 105 1.21 × 105 - 4 4 4 4 4 4 4 NO 3 ( NaNO3) 7.29 × 10 5.83 × 10 4.37 × 10 7.29 × 10 4.37 × 10 2.91 × 10 1.45 × 10 - 4 4 4 4 4 4 4 CH 3COO (CH3COONa) 7.19 × 10 5.75 × 10 5.75 × 10 4.31 × 10 2.87 × 10 1.43 × 10 2.87 × 10 2- 4 4 4 4 4 4 4 CO3 ( Na2CO3) 5.66 × 10 4.52 × 10 5.66 × 10 4.52 × 10 3.39 × 10 2.26 × 10 1.13 × 10 2- 4 4 4 4 4 4 4 SO 4 ( Na2SO4) 6.76 × 10 5.40 × 10 5.40 × 10 4.05 × 10 2.70 × 10 1.35 × 10 1.35 × 10 3- 3 3 3 3 3 3 3 PO 4 ( Na3PO4) 5.79 × 10 4.63 × 10 4.63 × 10 5.79 × 10 3.47 × 10 2.31 × 10 1.15 × 10 Citrate (Sodium citrate) 3.00 × 102 2.80 × 102 2.60 × 102 2.60 × 102 2.20 × 102 2.00 × 102 2.00 × 102 2 2 2 2 2 2 2 Oxalate ( Na2C2O4) 6.56 × 10 5.25 × 10 6.56 × 10 5.25 × 10 3.94 × 10 2.62× 10 1.31 × 10 Tartrate (NaKtartrate) 6.40 × 102 6.40 × 102 7.40 × 102 7.40× 102 6.20 × 102 5.60 × 102 5.40 × 102 Fulvic acid 5.20 × 101 5.20 × 101 3.80 × 101 4.80 × 101 5.00× 101 3.20 × 101 3.20 × 101 Humic acid 9.80 × 101 9.80 × 101 8.00 × 101 8.60 × 101 8.20 × 101 4.60 × 101 4.60 × 101

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Preconcentration factor and breakthrough capacity The preconcentration limit was determined by increasing the volume of metal ion solution and keeping the amount of loaded metal ion constant at 10 μg. The maximum preconcentration factors achieved for Cu(II), Co(II), Ni(II), Zn(II), Cr(III), Cd(II), and Pb(II) were 440, 380, 380, 360, 280, 280 and 260 with the corresponding preconcentration limit of 4.54, 5.25, 5.25, 5.55, 7.14, 7.14 and 7.69 µg L-1 respectively. Breakthrough capacities (Table 5.5) are more significant and useful than overall sorption of the resin in the column. capacities when working under dynamic condition, as it gives the actual working capacity.

Table 5.5 Preconcentration and breakthrough profiles of metal ions (Column parameters: 5.0 mL min-1, sorption flow rate; 3.0 mL min-1, elution flow rate; 0.5 g, resin) Overall sorption Breakthrough Breakthrough Degree of Metal Total volume capacity Capacity Volume column ions (mL) (µmol g-1) (µmol g-1) (mL) utilization Cu(II) 2600 787.45 692.91 2200 0.88 Co(II) 2250 746.53 641.83 1900 0.86 Ni(III) 2250 728.46 629.32 1900 0.86 Zn(II) 2150 643.18 551.38 1800 0.86 Cr(III) 1700 624.82 531.72 1400 0.85 Cd(II) 1700 295.59 249.11 1400 0.84 Pb(II) 1650 152.63 125.36 1300 0.82

The breakthrough volume, which corresponds to the volume at which the effluent concentration of any chemical species from the column is about 3-5% of the influent concentration, was determined by applying the recommended procedure to 1650-2600 mL of the metal ion solution. The effluent fractions were collected in 5 mL and analyzed for the presence of the metal. The overall capacity, breakthrough capacity and the degree of utilization was determined by the literature method [49]. The total sorption capacity calculated on the basis of total saturation volume was compared with the corresponding breakthrough capacities (Figure 5.9) for each metal. The closeness of the dynamic capacity to the total sorption capacity and the high preconcentration factors reflect the applicability of the column technique for preconcentration.

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Figure 5.9 Breakthrough curves for sorption of metal ions: C/Co is the concentration ratio of the effluent to influent. (Column parameters: 4 mL min-1, sorption flow rate; 0.5 g, resin)

5.4 Analytical figures of merit The accuracy of the present method was evaluated from the results of the analysis of various SRMs including environmental, biological and alloy samples applying recommended column procedure. Calculated Student’s t (t-test) values for respective metal ions were found to be less than the critical Student’s t-value of 2.78 at 95% confidence level for N=5 (Table 5.6). Hence, the mean values were not statistically significant from the certified values indicating that the method could be applied successfully for the analysis of real samples constituting different matrices.

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Table 5.6 Analysis of standard reference materials for metal ion contents (Column parameters: 5.0 mL min-1, sorption flow rate; 3.0 mL min-1, elution flow rate; 0.2 g, resin) Samples Certified value Found by proposed method Calculated (µg g-1) (µg g-1 )a Student's t Vehicle Co: 3.3, Cu: 67.0, Ni: Co:3.25(4.4), Cu:64.8(3.6), Ni:17.6 0.78, 2.10, exhaust 18.5, Zn: 1040.0, Pb: (4.6) , Zn:1028.1(1.5), 2.48, 1.71, particulates 219, Cr: 25.5, Cd: 1.1 Pb:209.5(3.8), Cr:24.3(4.5), Cd: 2.66, 2.45, NIES 8 c 1.1(4.5) 2.21 Pond Zn: 343, Ni: 40, Co: 27, Zn:333.2(2.9), Ni:37.9(4.5) , Co: 2.26, 2.75, sediment Cu: 210, Cr: 75 25.6(4.6), Cu:201.8(3.4), Cr: 2.65, 2.67, Chlorella Zn: 20.5, Cu: 3.5, Co: Zn:19.6(3.9), Cu:3.4(3.6), 2.63, 1.82,

NIES 3 0.87, Pb: 0.6 Co: 0.83(4.7), Pb:0.57(4.8) 2.29, 2.45 Human hair Zn: 169.0, Cu: 16.3, Ni: Zn:161.4(3.9), Cu:15.6(3.9), 2.70, 2.57, NIES 5c 1.8, Co: 1000, Ni:1.7(4.8), Co:986.3(1.4), 2.74, 2.21, Pb: 6 Pb:5.7(4.5) 2.61 Tea leaves Zn:33.0, Cu: 7.0, Zn:31.6(4.1), Cu:6.7(4.3), 2.41, 2.32, NIES 7c Ni:6.5, Cd: 300 Ni:6.2(4.5), Cd:289.5(3.1) 2.40, 2.61

Rompin Cu: 640, Zn: 1030.0, Cu:625.5(2.1), Zn:1018.3(1.2), 2.46, 2.14, hematite, Pb: 210 Pb:201.7(3.4) 2.70 JSS (800-3) d

Zinc base die- Cu: 1320.0, Ni: 29.0, Cu:1283.0(2.7), Ni:27.5 (4.6), 2.43, 2.63, casting alloy Pb: 82, Cr: 36, Cd: 51 Pb:78.5(3.9), Cr:34.3(4.4), 2.55, 2.51,

C NBS 627 e Cd:48.3(4.6) 2.71

a RSD, = Relative standard deviation, N=5 ; b at 95 % confidence level; c National Institute of Environmental studies (NIES); d Iron and

Steel institute of Japan (JSS) e National bureau of Standards (NBS)

Using optimum conditions, the precision of the method was evaluated. Six successive sorption and elution cycles of 10 µg of each metal ion taken in 100 mL (eluted in 5 mL of 4 mol L-1 HCl) were performed following the recommended procedure. It was found that the mean percentage recoveries of all the metal ions studied were 97.5-101.4% at 95 % confidence level. The RSD values were calculated to be below 5 %. The results of water analysis with RSD < 5 % support the applicability of the method.

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A blank run was also performed applying recommended column procedure with 100 mL of aqueous solution prepared by adding suitable buffer (excluding metal ions) and finally eluting the same in 5 mL before subjecting it to FAAS determination. The detection limits, evaluated as three times the standard deviation (s) of the blank signal, were found to be 0.56, 0.65, 0.64, 0.70, 0.75, 0.88, and 1.17 µg L-1 for Cu(II), Co(II), Ni(II), Zn(II), Cr(III), Cd(II), and Pb(II) respectively.

5.5 Applications

Determination of metal ions in natural water samples Applicability of the present method for preconcentration and determination of metal ions was accomplished by analyzing 500 mL of each water sample following recommended column procedure (direct method). The metal ion determinations were also confirmed from various real water (500 mL) samples which were spiked with known amount (2.5 µg) of individual metal ions. Recommended column procedure was then applied to determine the total metal ion contents. The close agreement of the results found for each metal ion in or spiked (added) to the various water samples (Table 5.7) indicates the reliability of the present method for metal analyses in water samples of various matrices without significant interference (recoveries ≥ 95%) .

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Table 5.7 Preconcentration and determination of metal ions in natural waters collected from various locations (Column parameter: 500 mL, solution; 5.0 mL min-1, sorption flow rate; 3.0 mL min-1, elution flow rate; 0.5 g, resin) -1 a c Analysis of analyte ions in real samples µg L ±confidence limit (% Recovery) Cu(II) Co(II) Ni(II) Zn(II) Cr(III) Cd(II) Pb(II) Samples Added Found Added Found Added Found Added Found Added Found Added Found Added Found

µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 3.47± 0.29 0 6.72±0.38 0 5.23±0.40 0 8.29±0.72 0 3.94±0.36 0 4.26±0.34 0 N.D.b 0 Tap water 11.95±0.80 10.15±0.63 13.33±1.15 8.92±0.46 9.26±0.94 5.06±0.45 8.44± 0.81 5 5 5 5 5 5 5 (104.6) (98.4) (100.8) (99.6) (100) (101.2) (99.4)

0 13.25±0.59 0 7.02±0.41 0 17.95±1.41 0 5.98±0.41 0 6.64±0.51 0 N.D.b 0 4.33±0.36 Sewage water 18.30±1.04 11.98±0.92 22.95±1.82 10.99±0.49 11.57±1.06 5.12±0.52 9.24±0.82 5 5 5 5 5 5 5 (101) (99.2) (100) (100.2) (98.6) (102.4) (98.2) 0 9.17±0.47 0 9.41± 0.63 0 6.45±1.11 0 4.87±0.38 0 5.87±0.49 0 4.01±0.34 0 5.07±0.36 Canal 13.17±0.85 13.46± 0.70 11.33±0.84 9.95±0.46 10.80±1.12 9.09±0.85 10.07±0.85 water 5 5 5 5 5 5 5 (100) (101) (97.6) (101.4) (98.6) (101.6) (100)

0 17.86±0.84 0 12.99±0.74 0 11.31±1.16 0 8.63±0.62 0 9.61±0.66 0 7.62±0.62 0 7.15±0.69 River 22.98±1.31 17.90±1.11 16.31±1.13 13.78±0.68 14.50±0.79 12.68±1.13 12.27±1.24 water 5 5 5 5 5 5 5 (102.4) (98.2) (100) (103) (97.8) (101.2) (102.4) ts a Confidence limit, C.L = x ± , N=3 at 95% confidence level; b N.D.= not detected; c =% Recovery. N

156

Table 5.8 Analysis of Common carbs (Cyprinus carpio), mango pulp and mint leaves for metal content (Column parameter: 50 mL, solution; 5.0 mL min-1, sorption flow rate; 3.0 mL min-1, elution flow rate; 0.2 g, resin) -1 a c Metal ion found by proposed method µg L ±confidence limit (% Recovery) Cu(II) Co(II) Ni(II) Zn(II) Cr(III) Cd(II) Pb(II)

Samples Added Found Added Found Added Found Added Found Added Found Added Found Found Added -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 µg g µg g µg g µg g µg g µg g µg g µg g µg g µg g µg g µg g µg g-1 µg g

0 0.77±0.07 0 N.D.b 0 N.D.b 0 1.32±0.07 0 0.17±0.01 0 N.D.b 0 0.37±0.03 Muscles 1.79±0.16 1.04±0.10 0.99±0.11 2.32±0.16 1.15±0.11 1.03±0.11 1.34±0.12 1 1 1 1 1 1 1 (102) (104) (99) (100) (98) (103) (97) 0 0.19± 0.02 0 N.D.b 0 N.D.b 0 1.96±0.09 0 0.35± 0.03 0 0.42±0.03 0 0.47±0.03 Livers 1.20±0.12 1.03±0.10 0.98±1.11 2.99±0.21 1.37±0.13 1.42±0.11 1.44±0.11 1 1 1 1 1 1 1 (101) (103) (98) (103) (102) (100) (97) 0 0.52± 0.04 0 N.D.b 0 0.20± 0.02 0 2.14±0.11 0 0.40± 0.03 0 0.13±0.02 0 0.28±0.03 Gills 1.55±0.10 1.01± 0.11 1.23±0.12 3.15±0.21 1.40±0.12 1.10±0.12 1.27±0.13 1 1 1 1 1 1 1 (103) (101) (103) (101) (100) (97) (99) Mango 0 2.55±0.17 0 N.D.b 0 0.95±0.09 0 2.62±0.15 0 1.85±0.15 0 0.92±0.09 0 0.37±0.04 Pulp 3.55±0.27 1.02±0.09 1.99±0.16 3.60±0.22 2.82±0.20 1.96±0.13 1.37±0.10 1 1 1 1 1 1 1 (100) (102) (104) (98) (97) (104) (100) Mint 0 2.02±0.14 0 0.55±0.01 0 1.14±0.09 0 5.05±0.27 0 2.12±0.13 0 0.13±0.01 0 1.13±0.09 leaves 3.05±0.19 1.55±0.11 2.17±0.17 6.02±0.52 3.11±0.23 1.15±0.14 2.11±0.15 1 1 1 1 1 1 1 (103) (100) (103) (97) (99) (102) (98) ts a Confidence limit, C.L = x ± , N=3 at 95% confidence level; b N.D.= not detected; c =% Recovery. N

157

Analysis of Cyprinus carpio, mango pulp and mint leaves The analytical method was used for each of the metal ion determination in or added (1 µg) to Cyprinus carpio, Mango pulp and Pudhina were subjected to preconcentration according to the recommended column procedure after their pretreatment (digested in 50 mL). The results (Table 5.8) show that recovery could be made with a good precision of RSD < 5%.

5.6 Conclusion

This salicylanilide immobilized chelating resin coupled with FAAS offers a simple, accurate and sensitive method for preconcentration and determination of trace heavy metals in various matrices found in natural samples. The resin shows excellent ability for the exclusion of alkali and alkaline earth elements which otherwise often interfere in the subsequent FAAS determination of metals. The tolerance ratio for various naturally occurring complexing agents, such as citrate, oxalate, humic acid and fulvic acid were found to be high enough to allow efficient preconcentration of river, canal, tap and sewage water thereby making unnecessary any prior digestion. Moreover, the use of a column preconcentration technique allows for the assessment of low trace metal concentrations, even by less sensitive determination methods such as FAAS. The use of carcinogenic organic solvents as eluting agent in the proposed method is eliminated. AXAD-16-SALD is found to be superior to other sorbents, possessing similar ortho orientation of functional groups, in many aspects. The regenerability of this resin is better than o-aminophenol [50], pyrocatechol [51], thiosalicylic acid [52], salicylic acid [53]

immobilized Amberlite XAD-2 resins and nitrosonaphthol [54] immobilized Amberlite XAD-16 resin. The half-loading time is superior to o-aminobenzoic acid immobilized Amberlite XAD-4 [55] and other reported work [50]. The preconcentration limit is more favorable than previous works [50,51,56]. This work covers comprehensive characterization of the newly functionalized resin and reports validation of the method prior to its applications in the analysis of real samples comprising varying complex matrices. To our literature knowledge, no study is available on the use of Amberlite XAD-16 fixed with salicylanilide as sorbent for the preconcentration of trace metal ions from various media.

158

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Chapter 6

Flame atomic absorption spectrometric determination of trace metal ions in environmental and biological samples after preconcentration on a new chelating resin containing p-Aminobenzene Sulfonic Acid

6.1 Introduction

The use of chelating resins as sorbent, in solid phase extraction (SPE), is gaining popularity because of its salient features like selectivity, high enrichment factor and recovery, rapid phase separation, low cost, low consumption of organic solvents and the ability of combination with different detection techniques in the form of on-line or off-line mode [1]. The selective removal of toxic metal ions and recovery of precious metal ions in terms of environmental protection and economic consideration are of great significance [2-5]. Various chelating resins have been efficiently employed for the preconcentration and separation of trace elements from various real samples of environmental and biological origin prior to their determination by sophisticated analytical instruments [2,6-13]. Analytical instruments with flameless technique have fallen out of favor because they require more expensive equipment and are usually much more sensitive to interferences from matrix. Coupling of preconcentration method with flame atomic absorption spectrophotometry is usually preferred because of its simple detection system based on flame atomization [14-22]. The selectivity of a chelating resin may be improvised by considering the chelating ability of the immobilized multidentate ligands, nature and degree of cross-linking of a polymeric support [15,17,18,23-25], the pH of the solution and the kinds of the metal ions. Its hydrophobicity may be overcome by introducing hydrophilic functional groups so that the surface area of contact with aqueous phase metal ions is enhanced thereby facilitating efficient column operation [13,26-28]. The twin objectives of abundant chelating sites as well as their accessibility can be achieved by anchoring a ligand containing non-sterically hindered chelating moieties on to a polymeric support of high surface area and good porosity. The use of Amberlite XAD-16 resins, with its superior physical properties such as porosity, uniform pore size distribution and chemical stability among other Amberlite XAD series resins [26,27,29], has gained popularity as the polymeric support for the immobilization of chelating ligand of small molecular size. Therefore, it was thought worthwhile to functionalize Amberlite XAD-16, which has high surface area and excellent porosity, with a small-sized ligand such as p-aminobenzene sulfonic acid (with one amino group) by coupling through azo spacer (Figure 6.1). The presence of sulfonic group in the chelating moieties would enhance its hydrophilicity, which would lead to faster kinetics thereby decreasing the time required for the preconcentration of large volumes of samples of trace metal ions by column operation. High sorption capacity 164

could be expected due to the dual modes of ion exchange (with sulfonic group) and formation of coordination bonds (with free amine groups) for the retention of metal ions [30]. Recently method for trace analysis of cadmium on stannum/bismuth/poly(p-aminobenzene sulfonic acid) film electrode and complexation studies of Cu(II), Co(II), Pb(II) with 4-(2- hydroxybenzylideneamino) benzenesulfanilic acid have been studied [31,32]. An optimized procedure has been applied to the determination of trace metals in natural water samples, standard reference samples, multivitamin tablets, infant milk substitutes, hydrogenated oil, mango pulp, mint leaves and fish by flame atomic absorption spectrometry.

-CH-CH2-

a Mn+ N N NH2 b SO H 3 Figure 6.1 Structure of a monomeric unit of AXAD-16 modified with p-Aminobenzene sulfonic acid; (a) is the probable chelating site and (b) is the hydrophilic and ion exchange group that enhances the hydrophilicity and sorption capacity of the resin

6.2 Experimental

Both batch and column methods (Section 2.6) have been employed for the sorption and elution studies of Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III) and Pb(II) prior to their determination by flame atomic absorption spectrometry (FAAS). The recommended procedures (Section 2.5-2.6) have been applied for the determination of different experimental parameters, including physico-chemical properties, of the resin.

The physico-chemical properties that have been determined include:

165

The experimental parameters that have been optimized include:

 Optimum pH range for sorption was ascertained by applying the recommended procedure (Section 2.6.1)  Contact /half-loading time for sorption has been determined (Section 2.6.1)  Flow rate for sorption as well as for elution has also been determined (Section 2.6.2- 2.6.3)  Eluting agent for complete desorption (Section 2.6.3)  The recommended procedure (Section 2.6.2) was followed to determine the preconcentration factor  Breakthrough volume for sorption has been determined according to the recommended procedure (Section 2.6.2)  The recommended procedure (Section 2.6.2) was followed for the determination of limit of detection (LOD) for each metal.

The following experiments have been performed for the validation of the method:

The method has been applied for the following applications:

166

 Digestion of standard reference materials were carried out according to the recommended procedure as in section 2.3.3.  The recommended procedure (Section 2.3.4) was followed for the digestion of fish samples prior to their analysis for metal content.  The recommended procedure (Section 2.3.5) was followed for the digestion of mango pulp and mint leaves prior to their analysis for metal content.

6.3 Results and discussion

6.3.1 Characterization of the chelating resin Elemental analysis performed on the different products, formed at each step, to elemental analysis. The nitrogen content of the nitrated resin and the subsequent reduced product (aminated resin) was found to be 9.02 % and 11.15 %, respectively. Elemental analysis of AXAD-16-ABSA gave 53.92 %, 4.95 %, 12.65 % and 5.02 of C, H, N, and S respectively, which agrees well with the calculated values, considering the possible

stoichiometry of its repeat unit to be C14H13N3O3S.H2O (C, 55.08 %, H, 4.91 % N, 13.77 % and S, 5.24 %). The presence of 12.65 % of N suggests the incorporation of at least 3.0 mmol of p-Aminobenzene sulfonic acid if we assume that all the functional groups have been attached through azotisation.

In thermogravimetric analysis (Figure 6.2), the AXAD-16-ABSA resin shows an earlier weight loss of 6.05 % up to 150 °C. This initial step corresponds to the endothermic peak in the DTA curve which may be attributed to the loss of coordinated water molecule. The TGA and elemental analysis together suggest that at least one water molecules per repeat unit is sorbed. The subsequent exothermic peak corresponding to the weight loss of 10.82 % up to 220 °C may be due to the loss of functional groups. Thermal analysis indicated that the synthesized resin was stable up to 200 °C, above which degradation commences. The sorption capacity observed for the resin, which had been subjected to heating up to 220 °C before cooling to the required temperature, was found to reduce by 20 %.

167

Figure 6.2 TGA/DTA curves of AXAD-16-ABSA

The IR spectrum of the modified resin AXAD-16-ABSA was compared with that of untreated Amberlite XAD-16 (Figure 6.3). The additional bands at 3447, 1606, 1113, and −1 1026 cm may have appeared due to the incorporation of ABSA into the latter through azo

spacer. These bands may be assigned to the amino (–NH2), azo (–N=N–) and >S=O (last two) groups, respectively. The IR spectrum of the metal ion-free chelating resin was compared with those of the metal ion-saturated resin. A red shift (5-10 cm−1) in the bands of the azo group and the disappearance of the broadening of the amino group in the metal ion- saturated AXAD-16-ABSA suggest that these groups are involved in the chelation of metal ions whereby metal sorption takes place. The resin showed no loss in sorption capacity up to -1 a concentration of 5 mol L of acids, such as HCl, HNO3 and H2SO4, and alkaline medium constituting 3 mol L-1 NaOH. Hence, the resin exhibited high chemical stability. The water regain capacity was found to be 9.84 mmol g-1. This value reflects the high hydrophilicity of the resin which is satisfactory for column operation. The overall hydrogen ion capacity amounts to 6.56 mmol g-1 of resin, which may be contributed both by the sulfonic and amino groups present within the molecule. Theoretically, if 3.0 mmol of the reagent constituted per gram of the resin, the hydrogen ion capacity, due to the hydroxylic

168

group should have been 3.0 mmol g-1, which agrees well with the experimental value (2.75 -1 mmol g ) corresponding to hydrogen ion capacity with NaHCO3.

Figure 6.3 FT-IR spectrum of a) AXAD-16-ABSA and b) AXAD-16-ABSA saturated with Cu(II)

6.3.2 Optimum experimental parameters In order to optimize sorption of metal ions, the multivariate approach was followed to establish all the parameters. Each optimum condition was established by varying one of them and following the recommended procedure.

Effect of pH on sorption capacity Optimum pH of metal ion uptake was determined by static method by stirring excess of metal ions (100 µg mL-1) with resin for 120 min over a pH range of 2-10±0.01. Preliminary experiments showed that the maximum sorption of Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III), and Pb(II) (Figure 6.4) was observed at pH 4.0, 4.0, 6.0, 4.0, 4.0, 4.0, and 6.0, respectively. Hence, pH 4.0 for Cu(II), Ni(II), Co(II), Cr(III), Fe(III) and 6.0 for Zn(II) and Pb(II) were adjusted in all further experiments.

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Figure 6.4 Dependence of sorption capacity on the pH of the solution (Batch method parameter: 100 mL, solution; 100 µg mL-1, metal ions; 0.1 g, resin)

Kinetics of sorption To study the effect of time on the sorption, resin was stirred with solution containing

one of the metal ions (100 µg mL-1) at different temperatures for 2, 5, 10, 15, 20, 30, 60, 90

and 120 min (under the optimum conditions). The loading half time (t1/2) needed to reach 50

% of the total loading capacity was estimated from Figure 6.5 and reported in Table 6.1.

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Figure 6.5 Kinetics of sorption of metal ions on AXAD-16-ABSA (Batch method parameter: 100 mL, solution; 100 µg mL-1, metal ions; 0.1 g, resin)

Table 6.1 Kinetics and batch capacity of sorption of metal ions on AXAD-16-ABSA (experimental conditions:100 mL, solution; 0.1 g, resin) Batch Loading Rate constant ΔS/ Metal capacity/ -ΔH/ halftime k x10-2 /min -1 J mol-1 K-1± ions µmol g-1± J mol-1± S.D t1/2/min S.D S.Da Cu(II) 290.5±0.35 6.5 10.6 53.63±0.52 96.71±0.97 Ni(II) 255.0±0.36 7.0 9.8 51.54±0.48 92.36±0.58 Zn(II) 195.5±0.29 8.0 8.6 45.11±0.41 81.41±0.44 Co(II) 175.0±0.31 9.0 7.6 54.68±0.51 93.15±0.65 Cr(III) 150.2±0.38 11.0 6.2 62.95±0.46 104.03±0.55 Fe(III) 140.8±0.18 8.5 8.1 47.47±0.62 82.37±0.79 Pb(II) 95.5±0.26 16.5 4.0 76.32±0.68 123.23±0.88 a Standard Deviation (n=3)

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From the kinetics of sorption for each metal (Figure 6.5), it was observed that 20-30 min was enough for the sorbent to reach the saturation level for all the metals which reflects good accessibility of the chelating sites in the resin. The kinetic studies also showed that the temperature affected the rate constants significantly; that is, saturation was reached at a faster rate at higher temperature. This temperature effect may be a manifestation of the fact that the resin swells more completely at higher temperature, which allows metal ions to diffuse more easily into the interior of the resin, and that the sorption was an endothermic process and hence high temperature facilitates higher sorption. A plot (Figure 6.6) of log D versus 1/T, where the distribution ratio (D) represents the ratio of sorption capacity and the concentration of free metal ion at the equilibrium sorption, respectively, revealed that the distribution ratio increased with the increase of temperature.

Figure 6.6 Influence of temperature on the distribution ratio of metal ions (Batch method parameter: 100 mL, solution; 100 µg mL-1, metal ions; 0.1 g, resin)

This again implies that the sorption process was an endothermic process. The values of ΔH and ΔS were calculated (Table 6.1) using the slope and intercept from the above plots using the following relationship [25]: − ∆H ∆S log D = − + (1) 2.303RT 2.303R

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The dynamics of the adsorption process in terms of the order and the rate constant can be evaluated using the kinetic adsorption data. The process of the mention metal ions removal from an aqueous phase by any adsorbent can be explained by using kinetic models and examining the rate-controlling mechanism of the adsorption process such as chemical reaction, diffusion control and mass transfer. The kinetic parameters are useful in predicting the adsorption rate which can be used as important information in designing and modeling of the adsorption operation. The kinetics of removal of such ions is explicitly explained in the literature using pseudo first-order, second-order kinetic models [18]. k t Pseudo-first-order model: log(q − q ) =log q − 1 (2) e t e 2.303 t 1 t = + Pseudo-second-order model: 2 (3) qt k2 q e qe

Where k1 and k2 is the rate constant of pseudo first-order and pseudo-second- order -1 rate constant, respectively. qe and qt are adsorption capacity at equilibrium (mmol g ) and at anytime, respectively. The parameters obtained from the kinetics models are listed in Table 6.2.

Table 6.2 Kinetics parameter for sorption of different metal ions (experimental conditions: 100 mL, solution; 0.1 g, resin) Order of Parameters Cu(II) Ni(II) Zn(II) Co(II) Cr(III) Fe(III) Pb(II) reactions -1 K1 (min ) 0.122 0.133 0.175 0.116 0.087 0.128 0.060 Pseudo-first- -1 qe (mg g ) 16.15 12.38 10.47 10.63 6.19 5.12 22.05 order model R2 0.9866 0.9862 0.8614 0.9418 0.9622 0.9719 0.9497

K2 Pseudo- 0.0083 0.0086 0.0068 0.0065 0.0078 0.012 0.001 (g/mg.min)

second-order -1 qe (mg g ) 18.35 14.44 12.45 10.68 7.55 7.54 19.12 model R2 0.9998 0.9835 0.9995 0.9963 0.9941 0.9832 0.9902

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Figure 6.7a Pseudo first-order kinetic plots for removal of metal ions (Batch method parameter: 100 mL, solution; 100 µg mL-1, metal ions; 0.1 g, resin)

Figure 6.7b Pseudo second-order kinetic plots for removal of metal ions (Batch method parameter: 100 mL, solution; 100 µg mL-1, metal ions; 0.1 g, resin)

174

It is evident from Table 6.2 and Figure 6.7a and b that a more precise fit of kinetics

data was shown by the pseudo-second order model. The calculated qe values are closer to the experimental data than the calculated values of pseudo-first-order model and the values of regression coefficients (R2) are higher (0.99) than pseudo-first-order kinetic model.

Adsorption isotherm For an adsorption column, the column resin is composed of microbeads. Each binding particle immobilized to the micro bead can be assumed to bind in a 1:1 ratio with the solute sample passed through the column. At the concentration range (1.3 x 103 - 2.5 x 104 µg) studied for Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III), and Pb(II) the data were successfully applied for Langmuir isotherm. The Langmuir model assumes that sorption occurs on defined sites of the sorbent with no interaction between the sorbed species and that each site can accommodate only one molecule (monolayer adsorption) with the same enthalpy sorption, independent of surface coverage. The linearized form of Langmuir isotherm may be represented by the following equation [18].

Ce 1 Ce = + q q b q e m m (4) -1 Here, Ce is the equilibrium concentration (mg L ), qe is the amount adsorbed at -1 equilibrium (mg g ) and qm and b is Langmuir constants related to adsorption efficiency and energy of adsorption, respectively. The linear isotherm occurs when the solute concentration

is very small relative to the binding molecule of the solid phase. The linear plots of Ce/qe

versus Ce suggest the applicability of the Langmuir isotherms (Figure 6.8).

The values of qm and b were determined from slope and intercepts of the plots and are

presented in Table 5. From the values of adsorption efficiency, qm we can conclude that the maximum adsorption corresponds to a saturated monolayer of adsorbate molecules on adsorbent surface with constant energy and no transmission of adsorbate in the plane of the adsorbent surface. To confirm the favorability of the adsorption process, the separation factor

(RL) is calculated as presented in Table 6.3. The values were found to be between 0 and 1 which confirm the favorability of the adsorption process [17,18].

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Figure 6.8 Langmuir sorption isotherms depicting the sorption behaviors of metal ions onto AXAD-16-ABSA (Column parameters: 4 mL min-1, sorption flow rate; 0.1g, resin)

Table 6.3 Langmuir isotherm constants for sorption of metal ions (Column parameters: 4.0 mL min-1, sorption flow rate; 0.1 g, resin) Standard B qm 2 Metal ions RL R deviation (L mg-1) (mg g-1) (N=5) Cu(II) 0.9768 0.0661 18.25 0.9999 0.3572 Ni(II) 0.7088 0.3577 14.88 0.9994 0.4150 Zn(II) 0.9560 0.1196 12.55 0.9997 0.1936 Co(II) 0.9293 0.1321 10.21 0.9999 0.3404 Cr(III) 0.8740 0.3127 7.62 0.9993 0.2648 Fe(III) 0.6886 0.4865 7.74 0.9967 0.5348 Pb(II) 0.5359 0.6994 18.05 0.9992 0.8733

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Effect of flow rate for sorption and elution Optimum flow rate may be defined as the rate of flow of the effluent (or eluent) through the column at which more than 98 % sorption (or elution) metal ions takes place. The effect of flow rate on the sorption was studied by varying the flow rate 1-10 mL min−1 at the pH chosen for maximum sorption. Observations indicated that metal retention on the resin was optimum at a flow rate equal or lower than 5.0 mL min-1. Hence, a flow rate of 4.0 mL min-1 was maintained throughout the column operations. During the subsequent elution of the retained metals from the resin, recovery of higher than 98% was observed up to 2.0 mL min-1. However, 100 % recovery of the sorbed metals could be achieved up to a flow rate of 2 mL min-1 and hence, maintained for further studies. At higher flow rates, quantitative stripping of metal ions needed larger volumes of the eluent. The decrease in sorption, or exchange, with increasing flow rate may be due to the decrease in equilibration time between two phases. Such high flow rates for sorption as well as desorption support the superiority of the present resin over previously reported works [33].

Type of eluting agents and resin reusability test Quantitative elution of metal ions was studied with different mineral acids namely

H2SO4, HClO4, HCl and HNO3 using varying volumes (1-10 mL) and concentrations (1-5 -1 mol L ). Among the acids, H2SO4 and HClO4 could elute 90 % and 95 %, respectively, -1 -1 when a maximum of 10 mL (4.0 mol L ) each were used. When 10 mL of 2 mol L HNO3/2 mol L-1 HCl was used, almost complete desorption (> 99 %) was observed. The efficacy of -1 -1 the eluent (2 mol L HNO3/2 mol L HCl) was studied taking its different volumes (1-10 mL). It was found that 3-5 mL of acid was sufficient for quantitative elution (>99 %) of all the metal ions. The resin was subjected to several loading and elution cycles by the dynamic method. The resin bed can be regenerated fully with 2 mol L-1 HCl easily and can be regenerated fully up to 35 cycles, which is relatively higher than previous works [26, 34]. Therefore, multiple use of the resin is feasible. Similar results are shown by batch method also.

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Study of interferences Various cations and anions may interfere in the determination of metal ions through precipitate formation, redox reactions, or competing complexation reactions. In order to assess the analytical applicability of the resin, various common chemical species were checked for any interference. The effect of humic substances on metal-collection was also examined because both humic and fulvic acids are generally present in natural waters at µg mL-1 to ng mL-1 levels and form complexes with various heavy metals [35-37]. Very few literatures [38,39] have considered the interference of these humic substances on the preconcentration of trace metal ions from natural waters. The tolerance limit is defined as the ion concentration causing a relative error smaller than ± 5 % related to the preconcentration and determination of the analytes. Many anions and cations, which are inevitably associated with metal ions present at the trace level in all natural waters, produce no interference in the sorption of the heavy metals up to appreciable concentrations (Table 6.4).

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Table 6.4 Tolerance limit of foreign species (in binary mixtures) on sorption of metal ions (Column parameter: 100 mL, solution; 1 µg, metal ion; 0.1g, resin) Foreign species Tolerance ratio(Foreign species / Metal ion ) (µg L-1) Cu(II) Ni(II) Zn(II) Co(II) Cr(III) Fe(III) Pb(II) Na+ (NaCl) 3.53 × 105 2.75 × 105 2.75 × 105 1.96 × 105 1.57 × 105 1.17 × 105 0.38 × 105 K+ (KCl) 4.71 × 105 3.67 × 105 3.14 × 105 2.62 × 105 1.57 × 105 2.09 × 105 1.04 × 105 + 5 5 5 5 5 5 5 NH4 (NH4Cl) 2.67 × 10 2.08 × 10 2.67 × 10 2.08 × 10 1.48 × 10 1.48 × 10 1.49 × 10 2+ 4 4 4 4 4 4 4 Mg (MgCl2) 2.29 × 10 2.04 × 10 2.04 × 10 1.78 × 10 1.53 × 10 1.27 × 10 0.76 × 10 2+ 4 4 4 4 4 4 4 Ca (CaCl2) 2.16 × 10 1.80 × 10 1.80 × 10 1.44 × 10 1.08 × 10 0.72 × 10 0.72 × 10 Cl- (NaCl) 5.45 × 105 4.85 × 105 4.24 × 105 4.24 × 105 3.03 × 105 2.42 × 105 1.81 × 105 - 4 4 4 4 4 4 4 NO3 (NaNO3) 6.56 × 10 5.10 × 10 5.10 × 10 4.37 × 10 3.64 × 10 2.18 × 10 1.45 × 10 - 4 4 4 4 4 4 4 CH3COO (CH3COONa) 6.47 × 10 5.05 × 10 5.05 × 10 3.59 × 10 2.15 × 10 2.15 × 10 1.43 × 10 2- 4 4 4 4 4 4 4 CO3 (Na2CO3) 5.09 × 10 5.09 × 10 4.52 × 10 3.96 × 10 2.83 × 10 2.83 × 10 1.69 × 10 2- 4 4 4 4 4 4 4 SO4 (Na2SO4) 6.08 × 10 4.73 × 10 4.73 × 10 3.38 × 10 2.02 × 10 2.02 × 10 0.67 × 10 3- 3 3 3 3 3 3 3 PO4 (Na3PO4) 5.21 × 10 5.21 × 10 4.05 × 10 4.05 × 10 2.89 × 10 2.89 × 10 0.57 × 10 Citrate (Sodium citrate) 3.20 × 102 2.80 × 102 2.60 × 102 2.60 × 102 2.20 × 102 2.00 × 102 2.00 × 102 2 2 2 2 2 2 2 Oxalate(Na2C2O4) 5.91 × 10 5.91 × 10 4.59 × 10 4.59 × 10 3.28 × 10 2.62× 10 1.31 × 10 Tartrate (NaKtartrate) 5.40 × 102 5.40 × 102 6.40 × 102 6.40× 102 6.00 × 102 5.20 × 102 5.20 × 102 Fulvic acid 5.20 × 101 5.20 × 101 3.80 × 101 4.80 × 101 5.00× 101 3.20 × 101 3.20 × 101 Humic acid 9.80 × 101 9.80 × 101 8.00 × 101 8.60 × 101 8.20 × 101 4.60 × 101 4.60 × 101

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Preconcentration factor and breakthrough capacity The preconcentration limit was determined by increasing the volume of metal ion solution and keeping the amount of loaded metal ion constant at 10 μg. The maximum preconcentration factors achieved for Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III), and Pb(II) were 360, 300, 300, 290, 180, 180 and 160, respectively with the corresponding preconcentration limit of 5.55, 6.66, 6.66, 6.89, 11.11, 11.11 and 12.50 µg L-1, respectively. Breakthrough capacities (Table 6.5) are more significant and useful than overall sorption capacities when working under dynamic condition, as it gives the actual working capacity of the resin in the column.

Table 6.5 Preconcentration and breakthrough profiles of metal ions (Column parameters: 4.0 mL min-1, sorption flow rate; 2.0 mL min-1, elution flow rate; 0.3 g, resin) Overall sorption Breakthrough Breakthrough Degree of Metal Total volume capacity Capacity Volume column ions (mL) (µmol g-1) (µmol g-1) (mL) utilization Cu(II) 2100 310.0 269.5 1800 0.88 Ni(II) 1900 270.0 238.0 1500 0.88 Zn(II) 1900 209.5 175.4 1500 0.84 Co(II) 1800 195.0 159.0 1450 0.82 Cr(III) 1300 166.0 137.2 900 0.82 Fe(III) 1300 148.5 120.5 900 0.81 Pb(II) 1200 103.2 78.8 800 0.77

The breakthrough volume, which corresponds to the volume at which the effluent concentration of any chemical species from the column is about 3-5 % of the influent concentration, was determined by applying the recommended procedure to varying volume of the metal ion solution. The effluent fractions were collected in 5 mL and analyzed for the presence of the metal. The overall capacity, breakthrough capacity and the degree of utilization was determined by the literature method [40]. The total sorption capacity calculated on the basis of total saturation volume (Figure 6.9) was compared with the corresponding breakthrough capacities (Table 6.5) for each metal. The closeness of the dynamic capacity to the total sorption capacity and the high preconcentration factors reflect the applicability of the column technique for preconcentration. 180

Figure 6.9 Breakthrough curves for sorption of metal ions: C/Co is the concentration ratio of the effluent to influent (Column parameters: 4 mL min-1, sorption flow rate; 0.3 g, resin)

6.4 Analytical figures of merit

The accuracy of the present method was evaluated from the results of the analysis of various SRMs by applying recommended column procedure. Calculated Student’s t (t-test) values for respective metal ions were found to be less than the critical Student’s t-value of 2.78 at 95 % confidence level for N=5 (Table 6.6). Hence, the mean values were not statistically significant from the certified values indicating that the method could be applied successfully for the analysis of real samples constituting different matrices. Using optimum conditions, the precision of the method was evaluated. Six successive sorption and elution cycles of 10 µg of each metal ion taken in 100 mL (eluted in 5 mL of 2 mol L-1 HCl) were performed following the recommended procedure. It was found that the mean percentage recoveries of all the metal ions studied were 97.0-104.0 % at 95 % confidence level. The RSD values were calculated to be below 5 %. The results of water analysis with RSD < 5 % support the applicability of the method.

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Table 6.6 Analysis of SRMs, multivitamin tablets and food samples for metal ion contents (Column parameters: 4.0 mL min-1, sorption flow rate; 2.0 mL min-1, elution flow rate; Samples0 3 i ) Certified/Reported value Mean found by proposed method Calculated (µg g-1) µg g-1 (RSD )a Student's t value b Vehicle exhaust Co: 3.3, Cu: 67.0, Ni: Co:3.25(4.4), Cu:63.9(4.3), Ni:17.7 0.78, 2.52, 2.19, particulates 18.5, Zn: 1040.0, Pb: (4.6) , Zn:1025.2(1.8), Pb:210.8(3.6), 1.79, 2.41, 2.22 NIES 8 c 219, Cr: 25.5 Cr:24.5(4.1) Pond sediment Zn: 343, Ni: 40, Co: 27, Zn:331.9(3.2), Ni:38.1(4.3) , Co: 2.33, 2.59, 2.57, NIES 2 Cu: 210, Cr: 75 25.7(4.4), Cu:203.3(3.1), Cr: 71.4(4.3) 2.37, 2.62 Chlorella Zn: 20.5, Cu: 3.5, Co: Zn:19.8(3.9), Cu:3.4(3.3), Co: 2.02, 1.99, 2.29, NIES 3 0.87, Pb: 0.6, Fe: 1850 0.83(4.7), Pb:0.57(4.8), Fe:1820(2.4) 2.45, 1.53 Human hair Zn: 169.0, Cu: 16.3, Ni: Zn:162.1(3.7), Cu:15.5(4.2), 2.57, 2.74, 2.74, NIES 5c 1.8, Co: 1000, Pb: 6, Ni:1.7(4.8), Co:981.3(2.1), Pb:5.7(4.5), 2.02, 2.61, 2.45 Fe:225 Fe:215.3(4.1) Tea leaves Zn:33.0, Cu: 7.0, Ni:6.5 Zn:31.4(4.3), Cu:6.6(5.0), Ni:6.2(4.5) 2.65, 2.71, 2.40 NIES 7c Rompin Cu: 640, Zn: 1030.0, Pb: Cu:622.9(2.5), Zn:1017.6(1.4), 2.45, 1.94, 2.55 hematite, 210 Pb:202.6(3.2) JSS (800-3) d Zinc base die- Cu: 1320.0, Ni: 29.0, Pb: Cu:1295.0(1.9), Ni:27.5 (4.5), 2.27, 2.71, 2.61, casting alloy C 82, Cr: 36, Fe: 230 Pb:77.9(4.5), Cr:34.4(4.4), 2.36, 2.77

NBS 627 e Fe :220.7(3.4) Maxirich (Cipla) Cu: 398.2; Zn: 442.5 Cu: 385.6 (3.6), Zn: 431.2 (3.4) 2.03, 1.72

Lactogen Cu: 2.9; Zn: 37.0 Cu: 2.80 (3.1), Zn: 35.4 (3.8) 2.57, 2.66 1(Nestle) Vanaspati ghee Ni: 0.45 Ni: 0.44 (5.0) 1.01 a RSD, = Relative standard deviation (%), N=5 ; b at 95 % confidence level; c National Institute of Environmental studies (NIES); d Iron and Steel institute of Japan (JSS) ; e National bureau of Standards (NBS),

A blank run was also performed applying recommended column procedure with 100 mL of aqueous solution prepared by adding suitable buffer (excluding metal ions) and finally eluting the same in 5 mL before subjecting it to FAAS determination. The detection limits, evaluated as three times the standard deviation (s) of the blank signal, were found to be 0.72, 0.89, 1.05, 0.98, 1.17, 0.69 and 1.91 µg L-1 for Cu(II), Ni(II), Zn(II), Co(II), Cr(III), Fe(III), and Pb(II) respectively. [16,18,24]

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6.5 Applications

Determination of metal ions in natural water samples Applicability of the present method for preconcentration and determination of metal ions was accomplished by analyzing 500 mL of each water sample following recommended column procedure (direct method). The metal ion determinations were also confirmed from various real water samples (500 mL), which were spiked with known amount (2.5 µg) of individual metal ions. Recommended column procedure was then applied to determine the total metal ion contents. The close agreement of the results found for each metal ion in or spiked (added) to the various water samples (Table 6.7) indicates the reliability of the present method for metal analyses in water samples of various matrices without significant interference (recoveries ≥ 95 %).

Analysis of real samples of complex matrices The recommended column procedure was applied for the analysis of multivitamin tablets, infant milk substitute and hydrogenated oil (Table 6.6), mango pulp, mint leaves and fish after their pretreatment (digested in 50 mL). The results (Table 6.8) show that recovery could be made with a good precision of RSD < 5 %).

183

Table 6.7 Preconcentration and determination of metal ions in natural waters collected from various locations (Column parameter: 500 mL, solution; 4.0 mL min-1, sorption flow rate; 2.0 mL min-1, elution flow rate; 0.3g, resin) -1 a b Metal ion found by proposed method, µg L ±confidence limit (% Recovery) Cu(II) Ni(II) Zn(II) Co(II) Cr(III) Fe(III) Pb(II) Samples Added Found Added Found Added Found Added Found Added Found Added Found Added Found

µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1 µg L-1

Tap 0 5.50±0.35 0 6.08±0.43 0 3.58±0.32 0 4.30±0.35 0 4.40±0.36 0 5.40±0.52 0 2.95± 0.27 Water 5 10.70±0.41 5 11.12±0.58 5 8.51±0.22 5 9.22±0.32 5 9.36±0.47 5 10.60±0.50 5 8.04± 0.29 (104.0) (100.8) (98.6) (98.4) (99.2) (104.0) (101.2) Sewage 0 11.33±0.77 0 12.58±0.75 0 6.25±0.40 0 7.02±0.47 0 5.70±0.46 0 8.66±1.01 0 3.80±0.35 Water 5 16.57±0.77 5 17.69±0.94 5 11.34±0.33 5 12.15±0.58 5 10.57±0.53 5 13.55±0.87 5 8.92±0.33 (104.8) (102.2) (101.8) (102.6) (97.4) (97.8) (102.4) Canal 0 7.15±0.46 0 8.42± 0.46 0 4.50±0.41 0 6.60±0.47 0 5.30±0.47 0 4.21±0.36 0 5.29±0.43 water 5 12.10±0.54 5 13.33± 0.76 5 9.40±0.23 5 11.52±0.40 5 10.42±0.48 5 9.35±0.41 5 10.37±0.50 (99.0) (98.2) (98.0) (98.4) (102.4) (102.8) (101.6) River 0 9.96±0.61 0 8.20±0.50 0 6.24±0.37 0 7.80±0.54 0 5.59±0.46 0 7.62±0.62 0 4.35±0.42 Water 5 15.10±0.65 5 13.31±0.78 5 11.18±0.35 5 12.90±0.56 5 10.50±0.36 5 12.68±0.68 5 9.27±0.46 (102.8) (102.2) (98.8) (102.0) (98.2) (101.2) (98.4) ts a Confidence limit, C.L = x ± , N=3 at 95% confidence level; b % Recovery. N

184

Table 6.8 Analysis of fish, mango pulp and mint leaves for metal content (Column parameter: 50 mL, solution; 4.0 mL min-1, sorption flow rate; 2.0 mL min-1, elution flow rate; 0.1 g, resin) -1 a c Metal ion found by proposed method, µg g ±confidence limit (% Recovery) Samples Cu(II) Ni(II) Zn(II) Co(II) Cr(III) Fe(III) Pb(II) Added Found Added Found Added Found Added Found Added Found Added Found Added Found µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1 µg g-1

Mango 0 2.18±0.17 0 1.05±0.10 0 2.69±0.18 0 N.D.b 0 1.73±0.15 0 1.62±0.15 0 0.32±0.03 Pulp 1 3.16±0.27 1 2.04±0.09 1 3.69±0.16 1 1.02±0.22 1 2.72±0.20 1 1.66±0.13 1 1.33±0.10 (98) (99) (100) (102) (99) (104) (101) Mint 0 2.11±0.17 0 1.06±0.09 0 5.41±0.32 0 0.08±0.01 0 2.02±0.17 0 4.53±0.32 0 0.83±0.08 leaves 1 3.09±0.19 1 1.07±0.11 1 6.40±0.17 1 1.10±0.52 1 3.01±0.23 1 5.50±0.14 1 1.81±0.15 (98) (101) (99) (102) (99) (97) (98) Muscles 0 0.67±0.06 0 N.D.b 0 1.19±0.08 0 N.D.b 0 0.18±0.02 0 0.11±0.01 0 0.29±0.03 1 1 1 1 1 1 1 1.32± 1.70±0.16 0.99±0.11 2.20±0.16 1.04±0.10 1.15±0.12 1.08±0.12 0.13 (103) (99) (101) (104) (97) (97) (103) Livers 0 0.22± 0.02 0 0.16±0.02 0 1.78±0.12 0 N.D.b 0 0.38± 0.03 0 0.42±0.03 0 0.42±0.04 1 1.25±0.13 1 1.20±0.13 1 2.82±0.20 1 1.01±0.10 1 1.37±0.12 1 1.42±0.11 1 1.44±0.13 (103) (104) (102) (101) (99) (100) (102) Gills 0 0.50± 0.04 0 0.25± 0.02 0 2.02±0.14 0 0.09±0.01 0 0.37± 0.03 0 0.12±0.01 0 0.24±0.03 1 1.51±0.14 1 1.23±0.13 1 3.05±0.25 1 1.01± 0.11 1 1.40±0.12 1 1.10±0.12 1 1.27±0.12 (101) (98) (103) (102) (103) (98) (103) ts a Confidence limit, C.L = x ± , N=3 at 95% confidence level; b N.D.= not detected; c =% Recovery. N

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6.6 Conclusion

This newly synthesized chelating resin coupled with FAAS offers a simple, accurate and sensitive method for preconcentration and determination of trace heavy metals in various matrices found in natural samples. The resin shows excellent ability for the exclusion of alkali and alkaline earth elements which otherwise often interfere in the subsequent FAAS determination of metals. The tolerance ratio for various naturally occurring complexing agents, such as citrate, oxalate, humic acid and fulvic acid were found to be high enough to allow efficient preconcentration of river, canal, tap and sewage water thereby making unnecessary any prior digestion. Moreover, the use of a column preconcentration technique allows for the assessment of low trace metal concentrations, even by less sensitive determination methods such as FAAS. The use of carcinogenic organic solvents as eluting agent in the proposed method is eliminated.

This work covers comprehensive characterization of the newly functionalized resin and reports validation of the method prior to its applications in the analysis of real samples comprising varying complex matrices. The results obtained demonstrated good reproducibility. Overall, AXAD-16-ABSA has a higher sorption capacity, [33,41,42] preconcentration factor [33,42,43], preconcentration limit [33,41], detection limit [33,42] and tolerance limits [33] which are superior in comparison to previous works. To our literature knowledge, no study is available on the use of Amberlite XAD-16 fixed with p- Aminobenzene sulfonic acid as sorbent for the preconcentration of trace metal ions from various media.

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