Miniaturized Analytical Devices: Materials and Technology [1 ed.] 3527347585, 9783527347582

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Miniaturized Analytical Devices

Miniaturized Analytical Devices Materials and Technology

Edited by Suresh Kumar Kailasa and Chaudhery Mustansar Hussain

Editors Prof. Suresh Kumar Kailasa

Department of Chemistry Sardar Vallabhbhai National Institute of Technology (SVNIT) Ichchhanath Surat-Dumas Road 395007 Surat Gujarat India

All books published by WILEY-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for

Prof. Chaudhery Mustansar Hussain

British Library Cataloguing-in-Publication Data

New Jersey Institute of Technology Department of Chemistry & Environmental Sciences Newark New Jersey United States

A catalogue record for this book is available from the British Library.

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Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at . © 2022 WILEY-VCH GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Print ISBN: 978-3-527-34758-2 ePDF ISBN: 978-3-527-82720-6 ePub ISBN: 978-3-527-82722-0 oBook ISBN: 978-3-527-82721-3 Typesetting Straive, Chennai, India Printing and Binding

Printed on acid-free paper 10 9 8 7 6 5 4 3 2 1

v

Contents

Section 1

1

1.1 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.4.1 1.2.4.2 1.2.4.3 1.2.4.4 1.3

2

2.1 2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5

Miniaturized Devices in Analytical and Bioanalytical Sciences 1

Miniaturized Capillary Electrophoresis for the Separation and Identification of Biomolecules 3 Suresh K. Kailasa, Vaibhavkumar N. Mehta, and Jigneshkumar V. Rohit Introduction 3 Brief Summary of MCE 4 Fabrication of Microfluidic Chips 4 Designing Microfluidic Channels 5 Electrophoretic Separation 6 Detectors 6 Capability of Microchip Electrophoresis for the Separation and Identification of Biomolecules 7 Detection of Cancer Biomarkers 8 Assays of Immune Disorders and Microbial Diseases by MCE 10 Assays of Biomarkers by MCE 11 Summary 14 Acknowledgments 14 References 14 Portable Nanomaterials Impregnated Paper-Based Sensors for Detection of Chemical Substances 21 Khemchand Dewangan and Kamlesh Shrivas Introduction 21 General Aspects of Nanomaterials 22 Synthesis of Nanomaterials 22 Solvothermal/Hydrothermal Technique 23 Reduction of Metal Salts 25 Microemulsion Techniques 25 Sol–Gel 25 Polyol Processes 25

vi

Contents

2.3.6 2.3.7 2.3.8 2.3.9 2.4 2.5 2.5.1 2.5.2 2.6 2.6.1 2.6.2 2.6.3 2.6.4 2.7 2.7.1 2.7.2 2.7.3 2.8

Coprecipitation 26 Thermal Decomposition of Metal–Organic Complex 26 Temperature-Programmed Reaction in the Presence of NH3 Gas 26 Urea as Nitrogen Source 27 Characterization of Nanomaterials 27 Paper Substrate and Functional Materials 29 Uniqueness of Paper Substrate 29 Functional Materials and Fabrication Methods 29 Different Types of Detection Methods 30 Colorimetric 31 Electrochemical 32 Fluorescence 32 Surface-Enhanced Raman Scattering (SERS) 33 Applications of Nanomaterial-Based Paper Sensors 33 Environmental Aspects 33 Clinical Aspects 35 Food Safety Aspects 36 Conclusion and Future Prospects 36 References 37

3

Miniaturized Analytical Technology in Agriculture 49 Vaibhavkumar N. Mehta, Vimalkumar S. Prajapati, and Jigneshkumar V. Rohit Introduction 49 Miniaturized Analytical Techniques for the Fungal Detection in Plants 51 Miniaturized Analytical Techniques for the Virus Detection in Plants 53 Miniaturized Analytical Techniques for the Bacterial Detection in Plants 61 Conclusion and Future Perspectives 65 References 66

3.1 3.2 3.3 3.4 3.5

4

4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3

Solvent Extraction Coupled with Gas Chromatography for the Analysis of Polycyclic Aromatic Hydrocarbons in Riverine Sediment and Surface Water of Subarnarekha River and Its Tributary, India 71 Balram Ambade, Shrikanta S. Sethi, Amit Kumar, and Tapan K. Sankar Introduction 71 Materials and Methods 72 Description of Study Area 72 Sampling and Pretreatment 72 Extraction and Cleanup of PAHs from Samples 74 Analysis 74 Quality Assurance 74 Results and Discussion 75

Contents

4.3.1 4.3.1.1 4.3.1.2 4.3.2 4.3.3 4.3.3.1 4.3.3.2 4.3.3.3 4.4

PAH Concentration in Water 75 PAHs Concentration in Subarnarekha Riverine Sediment 76 PAH Concentration in Kharkai Riverine Sediment 77 PAH Composition 78 Analysis for Sources of PAHs 79 Diagnostic Ratio 79 Principal Component Analysis 82 Potential Ecosystem Risk Assessment 83 Conclusions 85 Acknowledgments 86 References 86

5

Laboratory-on-a-Chip: A Multitasking Device 91 Mansi Mehta, Bhikhu More, Tanvi Tamakuwala, and Gaurav Shah Introduction 91 LOC in Multiplexing Microfabricated Devices 91 LOC in Integration 92 History 92 LOC Manufacturing Technologies 92 PDMS (Polydimethylsiloxane) 93 Thermopolymers 93 Glass 93 Silicon 93 Paper 93 Advantages of LOC Compared to Conventional Technologies 94 Low Cost 94 Easy Use 94 Reduction of Human Error 94 Less Sample Requirement 94 High Parallelization 94 Fast Response 94 Process Control and Sensitivity 95 Cost Effective 95 Limitations of LOC Compared to Conventional Technologies 95 Industrialization 95 Signal/Noise Ratio 95 Additional Requirements for Efficient Work 95 Ethics 95 Applications of LOC in Different Fields 95 LOC in Genomics 95 LOC in Post-Genome Era 96 LOC in Immunological Assay 96 Organ-on-a-Chip 98 LOC in Food Safety 98 LOC in Environmental Monitoring 99

5.1 5.1.1 5.1.2 5.2 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.4 5.4.1 5.4.2 5.4.3 5.4.4 5.4.5 5.4.6 5.4.7 5.4.8 5.5 5.5.1 5.5.2 5.5.3 5.5.4 5.6 5.6.1 5.6.2 5.6.3 5.6.4 5.6.5 5.6.6

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Contents

5.6.7 5.6.8 5.7 5.8

LOC in Cancer Diagnosis 99 LOC in COVID-19 Detection 100 Present Challenges 101 Conclusion and Future Perspectives 102 References 102

6

Microscopic Tools for Cell Imaging 105 Parveen Parasar and Vivek K. Singh Introduction 105 Microscopy – History and Development 106 Live-cell Imaging Microscopy 107 Fluorescent Microscopy 107 Principle 107 Photobleaching 107 Fluorescence Microscopy and Dynamics of Cellular Processes 108 Confocal Microscopy of Living Cells: General Approach 109 Minimizing Photodynamic Damage 109 Improving Photon Efficiency 110 Use of Antioxidants 110 Fluorescence Imaging Modalities 110 Light Sheet Fluorescence Microscopy (LSFM) 110 Phase-contrast Microscopy 111 Principle 111 Quantitative Phase-contrast Microscopy 112 Principle 112 Holotomography (HT) or Optical Diffraction Tomography 113 Principle 113 Other Considerations 114 Oil Immersion and Water Immersion Lenses 114 Dry Lenses 114 Photodamage of Cells 114 Specimen Environment 115 Improve S/N Ratio 115 Conclusions 115 References 116

6.1 6.2 6.2.1 6.2.2 6.2.2.1 6.2.2.2 6.2.2.3 6.2.2.4 6.2.2.5 6.2.2.6 6.2.2.7 6.2.3 6.2.3.1 6.2.4 6.2.4.1 6.2.5 6.2.5.1 6.2.6 6.2.6.1 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 6.4

Section 2

7

7.1 7.2

Functionalized Nanomaterial for Miniaturized Devices 121

Ionic Liquid–Assisted Single-Drop Microextraction: A Miniaturized Sample Preparation Tool for Various Analytes 123 Janardhan R. Koduru and Lakshmi P. Lingamdinne Introduction 123 Ionic Liquids 124

Contents

7.2.1 7.2.2 7.2.3 7.2.4 7.3 7.3.1 7.3.1.1 7.3.1.2 7.3.1.3 7.3.2 7.3.2.1 7.3.2.2 7.4

8

8.1 8.2 8.2.1 8.2.1.1 8.2.1.2 8.2.2 8.2.2.1 8.2.2.2 8.2.3 8.2.3.1 8.2.3.2 8.2.4 8.2.4.1 8.2.4.2 8.2.5 8.2.5.1 8.2.5.2 8.3 8.3.1 8.3.2 8.4

9

9.1

Background 124 Chemistry and Functionality of Ionic Liquids 124 Classification of ILs 125 Various Applications of Ionic Liquids (ILs) 128 Ionic Liquid–Assisted SDME for Analytes 129 Factors Influencing Ionic-Liquid-Assisted SDME 129 Vapor Pressure and Thermal Stability of ILs 129 The ILs are Liquids in a Broad Range 131 Viscosity and Surface Tension of ILs 131 ILs in SDME Coupled with Various Analytical Detectors for Analysis of Various Analytes 131 Analysis of Organic/Bioorganic Molecules 132 Inorganic Analysis 134 Conclusion and Future Prospects 141 References 142 Functionalized 2D Nanomaterials for Miniaturized Analytical Devices 153 Thang P. Nguyen Introduction 153 2D Nanomaterials 154 Graphene 154 Synthesis of Graphene 154 Characteristics and Applications of Graphene 156 Transition Metal Oxides 156 Synthesis Method 157 Characteristics and Applications of TMOs 157 Transition Metal Chalcogenides 159 Preparation of TMCs 159 Characteristics and Applications of TMCs 162 MXenes 163 MXene Preparation 163 Characteristics and Applications of MXenes 163 2D Metal–Organic Frameworks 164 Synthesis of 2D MOFs 165 Characteristics and Applications of MOFs 166 Functionalization Methodologies 167 Inorganic Doping Method 167 Functionalized Organic Functional Groups 168 Outlook 169 References 171 Functionalized Materials for Miniaturized Analytical Devices 181 Hani Nasser Abdelhamid Introduction 181

ix

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Contents

9.2 9.3 9.3.1 9.3.2 9.3.3 9.3.4 9.3.5 9.4 9.5 9.6

10

10.1 10.2 10.2.1 10.2.2 10.2.3 10.2.4 10.3 10.3.1 10.3.2 10.3.3 10.4

11 11.1 11.2

11.2.1 11.2.2 11.3 11.3.1

Miniaturized Devices 182 Miniaturized Devices for Analysis 183 Optical Devices 183 Electrochemical Methods 184 Magnetic Relaxation Switches (MRSw) Assays 184 Microfluidic Technology 185 Mass Spectrometry 186 Applications of Nanomaterials in Miniaturized Separation Techniques 187 Advantages, Disadvantages, and Challenges 187 Conclusions 188 Acknowledgments 189 References 189 Microvolume UV–Visible Spectrometry for Assaying of Pesticides 197 Jigneshkumar V. Rohit and Vaibhavkumar N. Mehta Introduction 197 Ag NP–Based Microvolume UV–Visible Spectrometry for Analysis of Pesticides 198 Analysis of Fungicides 199 Analysis of Herbicides 202 Analysis of Insecticides 202 Analysis of Other Pesticides 204 Au NP–based Microvolume UV–Visible Spectrometry for Analysis of Pesticides 205 Analysis of Fungicides 205 Analysis of Herbicides 205 Analysis of Insecticides 209 Summary 212 References 212 Miniaturized Liquid Extractions in MALDI–MS Analysis 219 Nazim Hasan and Shadma Tasneem Introduction 219 MALDI/SALDI–TOF–MS Instrumentation and Ionization Expected Mechanism Before Miniaturization of Liquid Extraction by Nanoparticles 221 MALDI–TOF–MS Techniques 221 Miniaturization-Based NPs in SALDI/MALDI–TOF–MS Application 224 Miniaturization of Metal Nanoparticles as Affinity Probe for SDME Via MALDI–TOF–MS 225 Affinity Probe of Functionalized Au and Ag Nanoparticles as SDME 225

Contents

11.3.2 11.4 11.4.1 11.4.2 11.5 11.5.1 11.5.2 11.5.3 11.5.3.1 11.5.3.2 11.5.3.3 11.6

12

12.1 12.2 12.3 12.3.1 12.3.2 12.3.3 12.3.4 12.4 12.5

13

13.1 13.2 13.2.1

Nanoparticles and Ionic Liquid (NP-IL) Hybrid Probes as SDME 227 Miniaturization of Nanoprobes for LLME Via MALDI–TOF–MS 228 Miniaturized Nanoparticles as LLME Enrichment Probes for Biomolecules 228 Miniaturized Nanoparticle-Based LLME Affinity Probes for Bacterial Proteins 229 Miniaturization of Nanomaterial Affinity Probes for Biomolecules Liquid Extraction 233 Metal Nanoparticle–Based Miniaturization Liquid Extraction Probes 234 Semiconductor Quantum Dots (QDs)-Based Miniaturization Liquid Extraction Probes in MALDI–TOF Analysis 239 Metal-Oxide Nanomaterial–Based Miniaturization Liquid Microextraction for MALDI–TOF–MS 241 Phosphopeptides Enrichment by Liquid Microextraction Analysis by MALDI–TOF–MS 241 Miniaturization of Metal-Oxide Nanoparticles for Bacterial Proteins Liquid Microextraction Analysis by MALDI–TOF–MS 243 Miniaturization Nanoarray-Based Biochips for Biomolecule Analysis by MALDI–MS 247 Conclusion 250 References 250 Mechanisms and Applications of Nanopriming: New Vista for Seed Germination 261 Karen P. Pachchigar, Darshan T. Dharajiya, Sumeet N. Jani, Jaykishan N. Songara, and Gaurav S. Dave Introduction to Agriculture and Green Nanotechnology 261 Nanopriming for Better Crop Germination 263 Anticipated Mechanisms Underlying Nanopriming: Plant Physiology and Molecular-Level Interactions 264 Imbibition and Vigorous Seedling Growth 265 Osmotic Adjustment and Membrane Dynamics 266 Antioxidant and ROS Signaling 267 Hormonal Crosstalk and Metabolic Flux 268 Current Status of Nanopriming 269 Conclusion 273 References 273 Nanotechnology for Environmental Pollution Detection and Remedies 279 Nishant Srivastava and Gourav Mishra Introduction 279 Nanotechnology for Environmental Monitoring and Diagnosis 280 Nanosensors for Water Contamination 280

xi

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Contents

13.2.2 13.2.3 13.2.4 13.3 13.3.1 13.3.2 13.4

Nanosensors for Air Pollution 282 Nanosensors for Soil Contamination 283 Nanobiosensors 284 Nanotechnology for Environmental Remediation 285 Photocatalysis Or Advanced Oxidation Process for Environmental Remediation 286 Nanocomposites and Nanodevices for Environmental Remediation 288 Conclusion 289 References 289 Index 295

1

Section 1 Miniaturized Devices in Analytical and Bioanalytical Sciences

3

1 Miniaturized Capillary Electrophoresis for the Separation and Identification of Biomolecules Suresh K. Kailasa 1 , Vaibhavkumar N. Mehta 2 , and Jigneshkumar V. Rohit 3 1 Department of Chemistry, Sardar Vallabhbhai National Institute of Technology, Surat-395007, Gujarat, India 2 ASPEE Shakilam Biotechnology Institute, Navsari Agricultural University, Surat-395007, Gujarat, India 3 Department of Chemistry, National Institute of Technology Srinagar, Hazratbal Kashmir 190006, Kashmir, India

1.1 Introduction Microchip capillary electrophoresis (MCE) is one of the efficient bioanalytical tools for rapid separation and detection of bioactive molecules with high separation resolution [1–3]. It has proven to be a prominent tool for identification of nucleic acids and proteins in food and clinical microbiology [2]. Separation of biomolecules is a key platform for quantitative and qualitative analysis of target biomolecules in biological matrices. For the first time, Manz’s group integrated a simple analytical procedure on a small glass chip for the separation and detection of target chemical species [4], collectively referred to as “lab-on-a-chip” or micro total analysis systems (μTAS). In this concept, MCE is included due to the separation mechanism in a microchip with very short channels. As a result, target molecules from the mixture are effectively separated by using high electric field strengths. MCE has been widely applied as a rapid separation tool in various fields of science, i.e. proteomics, genomics, biomarkers, and forensics [5–9]. These reviews reported that MCE has shown better performance for the separation of target analytes compared to traditional capillary electrophoresis. The MCE has successfully separated >30 000 proteins from a single cell [10]. In this chapter, we summarize the recent developments of MCE for separation and identification of nucleic acids and proteins from clinical and food samples. We briefly describe the history and role of MCE in clinical and food microbial research. A section is devoted to applications of MCE for separation and identification of nucleic acids, proteins, and biomarkers from clinical and food samples. The analytical features of MCE for rapid separation and detection of biomolecules are tabulated, which provides significant information to scientists to know potential advancements of MCE in molecular biology.

Miniaturized Analytical Devices: Materials and Technology, First Edition. Edited by Suresh Kumar Kailasa and Chaudhery Mustansar Hussain. © 2022 WILEY-VCH GmbH. Published 2022 by WILEY-VCH GmbH.

1 Miniaturized Capillary Electrophoresis for the Separation and Identification of Biomolecules

1.2 Brief Summary of MCE Generally, MCE consists of four core parts: microfluidic chip, electric field, separation, and detector. The electric field is applied for sample concentration and separation. Figure 1.1 displays a T-shaped microfluidic chip. The microfluidic chip contains few reservoirs such as sample and buffer reservoirs. These reservoirs should be filled with a background solution, and sieving gels and pipetting and syringe pumps are used for fluidic control. Once the microfluidic chip is set up with these parts, a high electric field is applied to the reservoirs (sample) to separate the target analytes. The detector is placed at the end of the separation channel, which results in registering the zones for separation and transmitting the data for signal processing unit, which generates an electropherogram. In this section, an overview of fabrication of microfluidic chips, sample preparation (on-microfluidic chip), separation, and analyte detection is given.

1.2.1

Fabrication of Microfluidic Chips

800

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400 450 500

350

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130-bp

300 (ii) Mixture of TCRγ+ Cμ 130-bp

200 (iii) Cμ 90-bp

Electric field (V/cm, dotted line)

90-bp

250 300

(i) 50-bp DNA ladder

100 150 200

50

550 600 650 700

0.2 RFU

2852

750

So far, several methods have been adopted for the fabrication of microfluidic chips, such as reactive ion etching, wet etching, photolithography, conventional

RFU

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100 (iv) TCRγ (v) Negative control

0 08

13

18

23

28

Migration time (s)

Figure 1.1 Separation and identification of amplified-PCR products of T-cell lymphoma. 50-bp DNA ladder (i); mixture of TCRγ and Cμ (ii); positive control Cμ(130-bp) (iii); TCRγ (90-bp) (iv); and negative control (v). The dotted linesrepresent the applied electric field. Figure reprinted from Ref. [25] with permission.

1.2 Brief Summary of MCE

machining, hot embossing, injection molding, soft lithography, in situ construction, laser ablation, and plasma etching [11, 12]. Silicon or glass is used as raw materials for the fabrication of microfluidic chips. Microfluidic electrophoresis chips consist of two reservoirs (sample and buffer) connected to the separation channel. A wide variety of materials including ceramics, glass, and polymers (poly(methyl methacrylate), cyclic olefin copolymers, polycarbonate, polystyrene, and fluorescent poly(p-xylylene) polymer (Parylene-C) have been used for preparation of microfluidic chips. Paper and fabric-based disposal chips have also received attention in MCE [7]. Electroosmotic flow (EOF) is generated in microfluidic chips as the reservoirs are filled with a background solution or electrolyte. Since EOF significantly obstructs separation, microfluidic chips are coated with various chemicals and hydrogels to suppress EOF.

1.2.2

Designing Microfluidic Channels

Crossed-channel and T-shaped microfluidic chips are widely used in MCE, and the microchip channel is connected perpendicularly to other channels (sample and buffer). Microfluidic chips are also prepared with different designs. For example, a microfluidic chip (Agilent Bioanalyzer chip) is prepared with 16 reservoirs, of which 12 are for sample reservoirs and four for references and reagents. Accordingly, MCE has been successfully applied for the analysis of various bioactive molecules with high precision and accuracy. Microfluidic chip channels with a width of 10–100 μm and a depth of 15–40 μm are considered the best design for separation of analytes. Also, separation channel area is designed to be 165 mm and 8 × 8 mm2 and the number of channels of microchips is increased to 12–384 for separation of nucleic acids and multiple genotyping (384) molecules with reduced time and increased accuracy [3]. Furthermore, microchips are designed with 8, 12, 16, 48, and 384 parallel channels for rapid and efficient separation of a wide variety of analytes. The electric field (voltage application) and hydrodynamic pressure are applied for sample injection in MCE. On-chip peristaltic pump is used for hydrodynamic injection [13]. Inkjet and array techniques are droplet injection systems, which provide high throughput and the sample injection volume ranges from nano- to picoliters [14]. As microfluidic chips are compact in size, electrokinetic injection is the preferred method of sample injection, where the injection volume of the sample is strongly dependent on the applied voltage and injection time [15]. Further, hydrodynamic injection system requires a pump or pipette, which limits its use in MCE. It is usually carried out by variations in pressure, vacuum, reservoir (sample waste), and fluid levels of sample. Electrokinetic injection system contains several injection modes, i.e. floating, gated, dynamic, and pinched. In the pinched injection mode, a voltage is applied at various channels including sample, buffer waste, and buffer reservoirs, and, as a result, the sample is injected into the channel junctions, and further enters the separation channels. Although the pinched injection mode is well illustrated and low volume of analyte plugs, it decreases the sensitivity due to sample plug. In the floating injection mode, which is similar to the pinched design, the potential is not required in the buffer and buffer waste reservoirs, increasing the sample load due TM

5

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1 Miniaturized Capillary Electrophoresis for the Separation and Identification of Biomolecules

to diffusion of the sample into the separation channel. Voltage can be applied at the sample and buffer reservoirs, and two waste reservoirs are grounded in this mode. The sample is injected into the separation channel by switching off the voltage at the reservoir (buffer), loading several amounts of the sample, which could help to improve the sensitivity of MCE. Then, the voltage is again switched on at the buffer reservoir. In dynamic injection mode, electroosmosis is required to inject the sample into separation channels, which can also improve the sample load.

1.2.3

Electrophoretic Separation

Target analytes are effectively separated in separation channels via electrophoretic separation by applying an electric field. Electrophoretic migration of analytes occurs due to the electric field and flow of liquid, collectively known as EOF. Various electrophoretic separations including electrokinetic chromatography, gel electrophoresis, and zone electrophoresis have been described in MCE [7]. Dielectrophoresis (DEP), gel electrophoresis, and zone electrophoresis modes are generally used in MCE. The DEP allows the separation of particles and cells by applying irregular electric fields (nonuniform) [7]. PC-3 human prostate cancer cells and polystyrene microbeads were separated by ionic liquid electrodes [16], and target analytes and particles were successfully separated via the on-chip procedure from human plasma [17]. The charged analytes migrate using the background solution (BGS) and are separated through electrophoretic mobility of target analytes, which leads to detection of analytes on the basis of descending mobility. As a result, small organic molecules including metabolites and drugs are effectively separated by the zone electrophoresis mode. Electrophoretic mobility of analytes takes place when an electric field is applied along a sieving matrix. The analytes migrate through the matrix, and the degree of migration is dependent on the size/weight of the analytes. Porous gels are used for the preparation of the sieving matrix, and the size of the gel mesh affects separation efficiency of MCE. Generally, separation efficiency of MCE will be greater with the increasing concentration of cross-linked gels. Viscosity of gels also increases with increasing concentration of the sieving matrix, making it difficult to load the gels into microfluidic channels. Common gels are prepared by using starch, agarose, cellulose, and polyacrylamide. Large biomolecules such as nucleic acids (DNA and RNA), proteins, and biomarkers can be successfully separated by using gel electrophoresis based on size-dependent separation. Polymerase chain reaction (PCR) is often used for the amplification of RNA and DNA to concentrate nucleic acids prior to their separation by MCE. Importantly, MCE has ability to separate base pairs with reduced time and improved resolution, exhibiting better analytical features compared to slab gel electrophoresis, which requires 1200 small- and medium-scale industries and has an SEZ named AIDA in Adityapur [21]. The first planned industrial city of India, Jamshedpur, is situated on the banks of Subarnarekha River and its main tributary Kharkai River [21]. These two rivers mainly flow through the Adityapur region of Jamshedpur where >1200 industries are operating, including iron, steel, chemicals, transport equipment, and cement, which can be the cause of water pollution in both rivers. The main concern of this study was about the PAH levels in Subarnarekha and Kharkai Rivers. Therefore, the study investigated the occurrence and levels of PAHs in the surface water and sediments of both rivers and the distribution of PAHs in their rural, town, and semi-town regions. Diagnostic ratios and principal component analysis (PCA) were used to estimate the distribution and identification of sources of PAH contamination.

4.2 Materials and Methods 4.2.1

Description of Study Area

The Subarnarekha River flows across three states – Jharkhand, Odisha, and West Bengal of India. Its source is located at 23∘ 18′ N, 85∘ 11′ E and the mouth is 21∘ 33′ N, 87∘ 23′ E. Subarnarekha passes through areas with extensive mining of copper and uranium ores, and as a result it is highly polluted. The tribal communities inhabiting the Chota Nagpur region and their livelihood are threatened by water pollution of the Subarnarekha River. The Kharkai River is the main tributary of the Subarnarekha River, the length of which is 136 km and the catchment area is 6.611 km2 . It flows through Adityapur, the highly industrialized area of Jamshedpur. It arises in Mayurbhanj district, Odisha, and after flowing past Rairangpur it heads north up to Saraikela and then toward the east, and finally it enters Subarnarekha in northwestern Jamshedpur. Iron ore is mined in the mountains of the headwaters of the Kharkai River and supplied to the steel plant in Jamshedpur. These two rivers mainly flow through the Adityapur region where many industries such as sponge iron, steel, chemicals, transport equipment, and cement are fully fledged operating. The study includes the characterization of the rivers in village, town, and semi-town areas. The comprehensive sampling sites are shown in Figure 4.1.

4.2.2

Sampling and Pretreatment

As shown in Figure 4.1, 56 sampling sites along the Subarnarekha and Kharkai Rivers were selected. To effectively monitor the distribution of pollution in both rivers, sampling sites were selected so that they ranged from low contaminated (low industrialization and urbanization) areas to heavily contaminated (highly

4.2 Materials and Methods

35 °N

India

Jharkhand

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20 °N

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4 3 Ranchi

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31. kumram 32. ichha 33. kesa 34. tatanagar 35. gobarsola 36. basangi 37. kalarada 38. ranipal 39. ateoani 40. rairangpur 41. sonari 42. bisira 43. manpita 44. kacha 45. jadugoda 46. mahulia 47. ghatsila 48. musabani 49. gagia 50. jharia 51. hatibari 52. gopiballabpur 53. rohini 54. navagram 55. raisol 56. dantan

50

Rairangpur Water sampling sites

1. batia 2. khiri 3. ketetoli 4. jorar 5. tati 6. rukka 7. turup 8. jumla 9. sosa 10. sakla 11. baranga 12. kotam 13. muri 14. koncha 15. dibadih 16. dulmi 17. suisa 18. chandil 19. khiri 20. rudia 21. burudih 22. manikui 23. adityapur 24. itagarh 25. samram 26. chapra 27. seraikela 28. gohira 29. kuri 30. charai

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53 54 55 56

Figure 4.1 Sampling sites of the Kharkai and Subarnarekha Rivers. PAH concentration in Subarnarekha sediments PAHs concentration in Kharkai sediments. PAH concentration in water samples

industrialized and urbanized) areas. A total of 112 samples, including 56 sediment samples and 56 water samples, were collected in the post-monsoon season of August 2018. Sediment samples were collected in polyethylene bags using a grab sampler, and water samples were collected directly from the river into 2 l glass jars. The samples were kept in the ice box and sent to the laboratory, and the sediment samples were kept in the refrigerator at −18∘ C before analysis. The water samples were stored in the refrigerator at –4 ∘ C before analysis. Furthermore, the water samples were carried for further extractions. All containers in contact with the sample were previously washed with diluted nitric acid and deionized water to eliminate the effect of other contaminants.

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4 Solvent Extraction Coupled with Gas Chromatography for the Analysis of Polycyclic

4.2.3

Extraction and Cleanup of PAHs from Samples

PAHs in sediment samples were extracted by solvent extraction procedure. The dry samples were collected, homogenized, properly sieved, and kept for the sample preparation. This process involved mixing of 2 g of dry sediment samples with 0.5 g anhydrous Na2 SO4 and using 10 ml of dichloromethane (DCM) for 1 h of ultra-sonication followed by centrifugation. Then, 3 ml of supernatant was filtered through 2 g of silica gel column with 11 ml 1 : 1(v/v) elution of hexane and DCM [22]. Solvent fractions were evaporated by exchanging acetonitrile with a final volume of 2 ml. PAH compounds were extracted from the water samples by the procedures mentioned in the study by Chakraborty et al. [23]. DCM was used for liquid–liquid extraction for three times (50, 25, and 25 ml) with 1 l of water samples. Then, the organic phase was separated and collected by mixing 10 g sodium chloride and shaking for 4–6 minutes for three times, and finally the combined organic phase was collected and the drying process started by passing it through sodium sulfate placed on glass wool of 3 cm and was concentrated using rotary evaporator. The PAHs labeled with Deuterium (Naphthalene-D8, Pyrene-D10, Phenanthrene-D10, Anthracene-D10, and Chrysene-D12) were used as surrogate standards. For sample extraction, a silica gel column of 30 cm long and 3 cm diameter was used; top of the column was filled with 10 g activated silica gel and 5 g anhydrous Na2 SO4 . The PAHs were eluted with 100 ml of DCM and n-hexane mixture (1 : 1, v/v). Finally, under a gentle stream of pure nitrogen, the eluent solvent was concentrated to 20 μl. To quantify all analytes, an internal standard hexamethyl benzene was added before analysis [12].

4.2.4

Analysis

The sample extracts of water and sediments of Subarnarekha and Kharkai Rivers were analyzed in an Agilent 7890B Gas Chromatograph (GC-FID) coupled with 5977A Mass Spectrometry using a HP-5 MS capillary column (30 m × 0.32 mm × 0.25 μm) with a 7 inch cage. In splitless mode, 1 μl of each sample was injected. High-purity nitrogen gas was used as carrier gas. The carrier gas was flowing at a flow rate of 1.83 ml/min. The initial oven temperature was set to 65 ∘ C for 3 minutes and then increased to 290 ∘ C at a rate of 5 ∘ C/min by holding for 20 minutes. The temperatures of injector and transfer line were set to 290 and 320 ∘ C, respectively. Using this technique, the 16 USEPA PAHs, Naphthalene (NA), Acenaphthene (AC), Acenaphthylene (ACY), Fluorene (Fluo), Phenanthrene (Phen), Anthracene (AN), Fluoranthene (Flur), Pyrene (Pye), Benzo(a)Anthracene (BaA), Chrysene (CHRY), Benzo(b)Fluoranthene (BbF), Benzo(k)Fluoranthene (BkF), Benzo(a)Pyrene(BaP), Dibenzo [a,h]anthracene (DBA), Benzo[g,h,i]perylene (BgP), and Indeno[1,2,3-cd]pyrene (IN) compounds were identified according to their retention time and mass spectra.

4.2.5

Quality Assurance

We applied the standard solution of 16 PAHs in acetonitrile (ID-3697900), deuterium-leveled PAHs, and hexamethyl benzene (CAS Number-87-85-4; Sigma

4.3 Results and Discussion

Aldrich, USA). The chemicals DCM (CAS Number-75-09-2), anhydrous Na2 SO4 (CAS Number-7757-82-6), sodium chloride (CAS Number-7647-14-5), and silica gel powder (CAS Number-112926-00-8) with AR grade were used in this study. The procedural blank samples, sample duplicates, samples spiked with surrogate standards Pyrene-D10 (CAS Number-1718-52-1), Anthracene-D10 (CAS Number1719-06-8), Phenanthrene-D10 (CAS Number-1517-22-2), Naphthalene-D8 (CAS Number-1146-65-2), and Chrysene-D12 (CAS Number-1719-03-5) were used as internal standards for the calibration of all species of PAHs, and the quality control testing for PAHs analysis relied on these methods. The recovery percentages of 16 PAHs in the water and sediment samples were approximately 90–105% and 80–95%, respectively. The standard deviation was