X Ray Fluorescence in Biological Sciences Principles Instrumentation and Applications Vivek K Singh All Chapter

X-Ray Fluorescence in Biological
Sciences: Principles, Instrumentation,

and Applications Vivek K. Singh
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X-Ray Fluorescence in Biological Sciences
X-Ray Fluorescence in Biological Sciences
Principles, Instrumentation, and Applications
Edited by
Vivek Kumar Singh

University of Lucknow
Lucknow, India
Jun Kawai
Kyoto University
Kyoto, Japan
Durgesh Kumar Tripathi

Amity University
Noida, Uttar Pradesh, India
This edition first published 2022
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v
Contents
List of Contributors xxiii

Preface xxxi
Part I General Introduction 1
1 X-Ray Fluorescence and Comparison with Other Analytical Methods (AAS, ICP-AES,
LA-ICP-MS, IC, LIBS, SEM-EDS, and XRD) 3
Kanishka Rawat, Neha Sharma, and Vivek Kumar Singh
1.1 Introduction 3
1.2 Analytical Capabilities of XRF and Micro-XRF 4
1.2.1 Micro-XRF 4
1.3 Comparison with Other Analytical Methods 4
1.3.1 Overview 4
1.3.2 Inductively Coupled Plasma (ICP) Analysis 5
1.3.2.1 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 5
1.3.3 Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) 10
1.3.4 Ion Chromatography (IC) 11
1.3.5 Laser-Induced Breakdown Spectroscopy (LIBS) 12
1.3.6 Proton-Induced X-Ray Emission (PIXE) 13
1.3.7 Scanning Electron Microscopy–Energy Dispersive X–Ray Spectroscopy (SEM-EDS) 14
1.3.7.1 Differences in XRF and SEM-EDS (Sample Handling, Experimental Conditions, Sample
Stress, and Excitation Sources) 14
1.3.7.2 Combination of SEM-EDS and μ-XRF 16
1.4 Comparison of XRF and XRD 17
1.5 Comparison of XRF and Raman Spectroscopy 18
1.6 Conclusion and Prospects 19
References 19
2 X-Ray Fluorescence for Multi-elemental Analysis of Vegetation Samples 21

Eva Marguí and Ignasi Queralt
2.1 Introduction 21
2.2 Features and Analytical Capabilities of XRF Configurations used in Vegetation Sample
Analysis 22
vi Contents
2.3 eneral Sample Treatment Procedures used for Vegetation Sample Analysis using XRF
G
Techniques 26
2.4 Applications of XRF in the Field of Vegetation Samples Analysis 29
2.4.1 Environmental Studies 29
2.4.2 Nutritional and Agronomic Studies 31
2.5 Concluding Remarks and Future Perspectives 32
References 33
3 X-Ray Fluorescence Studies of Tea and Coffee 37

Аnatoly G. Revenko and Darya S. Sharykina
3.1 Introduction 37
3.2 The Equipment Used 39
3.3 Preparation of Samples for Analysis 39
3.4 Examples of Practical Applications of XRF for Tea Research 40
3.5 Examples of Practical Applications of XRF for Coffee Research 46
3.6 Determination of the Elemental Composition of Krasnodar Tea Samples by
TXRF and WDXRF 50
3.6.1 Instrumentation 51
3.6.2 Suspension Preparation 51
3.6.3 Infusion Preparation 51
3.6.4 Acid Digestion 51
3.6.5 Preparation of Samples for WDXRF 52
3.6.6 Results and Discussion 52
3.7 Interelement Effects and Procedures of their Accounting 52
3.8 Conclusion 55
References 55
4 Total Reflection X-Ray Fluorescence and it’s Suitability for Biological Samples 61
N.L. Mishra and Sangita Dhara
4.1 Introduction 61
4.2 Advantages and Limitations of conventional XRF for Elemental Determinations in
Biological Systems 62
4.3 Factors Limiting the Application of XRF for Biological Sample Analysis 63
4.4 Modifying XRF to Make it Suitable for Elemental Determinations at Trace Levels:
Total Reflection X-Ray Fluorescence (TXRF) Spectrometry 63
4.4.1 Principles of TXRF 64
4.4.2 Theoretical Considerations 64
4.4.3 TXRF Instrumentation for Trace Element Determination 68
4.4.4 Sample Preparation for TXRF Analysis 68
4.5 Suitability of TXRF for Elemental Analysis in Biological Samples 70
References 72
Contents vii
5 Micro X-Ray Fluorescence and X-Ray Absorption near Edge Structure Analysis of Heavy
Metals in Micro-organism 73
Changling Lao, Liqiang Luo, Yating Shen, and Shuai Zhu
5.1 Introduction 73
5.2 Effects of Heavy Metals on Microbial Growth 73
5.3 Application of μ-XRF and XAS in Understanding the Cycling of Elements Driven by
Micro-organism 74
5.4 Application of μ-XRF and XAS in Understanding the Transformation of Elements
Driven by Micro-organisms 75
5.5 Application of μ-XRF and XAS in Understanding the Mechanism of Using
Micro-organisms in Bioremediation 76
5.6 The Advantage of Using μ-XRF and XAS to Explore the Interaction Mechanism Between
Micro-organisms and Heavy Metals 77
Acknowledgment 78
References 78
6 Use of Energy Dispersive X-Ray Fluorescence for Clinical Diagnosis 81

Yeasmin Nahar Jolly
6.1 Introduction 81
6.2 Determination of Arsenic Concentration in Human Scalp Hair for the Diagnosis
of Arsenicosis Disease 82
6.2.1 Background 82
6.2.2 Role of EDXRF 82
6.2.3 Collection and Preparation of Hair Sample 82
6.2.4 Sample Preparation 83
6.2.5 Sample Analysis 83
6.2.6 Accuracy and Precision of the Method 84
6.2.6.1 Construction of Calibration Curve 84
6.2.6.2 Measured Condition 84
6.3 Determination of Lead Concentrations in Human Whole Blood Using EDXRF
Technique with Special Emphasis on Evaluating Association of Blood Lead Levels with
Autism Spectrum Disorders (ASD) 85
6.3.1 Background 85
6.3.2 Role of EDXRF in Diagnosis of Blood Lead Level 86
6.3.3 Collection of Blood Sample and Preparation 87
6.3.4 Preparation of Pellets from Powdered Sample 87
6.3.5 Sample Irradiation 87
6.3.6 Precision and Accuracy of the Result 88
6.4 Conclusion 88
References 89
viii Contents
7 Preparation of Sample for X-Ray Fluorescence Analysis 91

Nuray Kup Aylikci, Onder Oruc, Ersin Bahceci, Abdelhalim Kahoul, Tolga Depci, and
Volkan Aylikci
7.1 Introduction 91
7.2 Solid Samples 92
7.2.1 Metallic Samples 93
7.3 Powder Samples 94
7.3.1 Grinding 95
7.3.2 Pelletizing 96
7.3.3 Fused Samples 97
7.4 Liquid Samples 98
7.5 Sample Preparation for Infinitely Thick and Intermediate Specimen 102
7.6 Sample Preparation of Animal Cells 105
7.7 Sample Preparation of Plant Section 106
7.8 Precautions During Sample Preparation and Handling 108
7.9 Conclusion and Future Directions 108
References 109
Part II Synchrotron Radiation XRF 115
8 Elemental Analysis Using Synchrotron Radiation X-Ray Fluorescence 117

M. K. Tiwari
8.1 Importance of Trace and Ultra-Trace Elemental Analysis 117
8.2 Various Methods for Trace Element Analysis 118
8.2.1 Atomic Absorption Spectroscopy (AAS) Method 118
8.2.2 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Method 119
8.2.3 Neutron Activation Analysis (NAA) Method 119
8.2.4 Accelerator Ion Beam Techniques 120
8.2.5 X-Ray Fluorescence (XRF) Method 120
8.2.6 Total Reflection X-Ray Fluorescence (TXRF) Method 121
8.3 Comparison of TXRF and EDXRF Geometries 122
8.4 Synchrotron Radiation 125
8.4.1 Selection of a Laboratory X-Ray Source for TXRF 126
8.5 Indus Synchrotron Radiation Facility 127
8.6 Microprobe X-Ray Fluorescence Beamline (BL-16) 128
8.6.1 Working Principles of a Double Crystal Monochromator (DCM) Optic 129
8.7 Experimental Facilities Available on the BL-16 132
8.7.1 Normal EDXRF Measurements 132
8.7.2 Total Reflection X-Ray Fluorescence (TXRF) Measurements 134
8.7.3 Elemental Quantification 137
8.7.4 X-Ray Fluorescence Analysis of Nanostructures 139
8.7.5 Microfocus X-Ray Beam Mode 141
Contents ix
8.7.6 Micro-Fluorescence Mapping 142

8.7.7 Micro-XRF Mapping Analysis of Old Archeological Tile Samples 143
8.8 Discussion and Summary 146
References 148
9 Synchrotron Radiation Based Micro X-Ray Fluorescence Spectroscopy of Plant

Materials 151
Katarina Vogel-Mikuš, Paula Pongrac, Peter Kump, Alojz Kodre, and Iztok Arc ̌on
9.1 Introduction 151
9.2 Instrumentation and Sample Preparation 152
9.3 Case Studies 154
9.3.1 Metal Tolerance Mechanisms in Hyperaccumulating Plants 154
9.3.2 Metal Toxicity and Tolerance in Plants and Fungi 155
9.3.3 Distribution of Mineral Nutrients and Potentially Toxic Elements in Grain 156
9.3.4 Investigation of Interactions between Plants and Engineered Nanomaterials 158
Acknowledgements 159
References 159
10 Micro X-Ray Fluorescence Analysis of Toxic Elements in Plants 163

Jian Liu and Liqiang Luo
10.2 Advantages of XRF Technique for Plants Analysis 163
10.3 Preparation of Plant Samples for μ-XRF Analysis 167
10.4 The Case Studies of Synchrotron μ-XRF for Determination of Toxic Elements in Plants 167
10.4.1 Applications in Edible Plants 168
10.4.2 Applications in Accumulating Plants 169
10.4.3 Applications in Hyperaccumulator Plants 169
10.4.4 The Case Studies of Laboratory μ-XRF to Determine Elements in Waterlogged
Oenanthe javanica DC 170
10.5 Conclusion and Outlook 171
References 172
11 Micro X-Ray Fluorescence Studies of Earthworm (Benthonic Fauna) in Soils and

Sediments 175
Jing Yuan and Liqiang Luo
11.2 Sample Preparation Methods 176
11.3 Earthworms and Soil Ecosystem 176
11.3.1 Case 1-Bioaccumulation of Arsenic (As) in Earthworms 177
11.3.2 Case 2-Silver(Ag) Nanoparticles Localization in Earthworms 178
11.4 Overview 178
References 180
x Contents
12 Synchronous Radiation X-Ray Fluorescence Analysis of Microelements in

Biopsy Tissues 183
V.A. Trunova
12.2 Samples Preparation 184
12.3 Materials and Methods 186
12.4 SRXRF Measurements 187
12.5 SRXRF Biopsy Material of Living Organisms 189
12.5.1 The Elemental Composition of Derivatives of Human Epithelial Tissues 189
12.5.2 Dynamics of Derivatives of Epithelial Tissues, Human Hair and Nails 191
12.5.2.1 Dynamics of Derivatives of Epithelial Tissues, Human Hair, and Nails 192
12.6 Study of Elemental Composition and Inter-Element Correlations in the Liver and Lungs
of Animals with Food Obesity 193
12.7 Concluding Remarks 199
References 199
Part III Total Reflection XRF 203
13 Total Reflection X-Ray Fluorescence Analysis of some Biological Samples 205

N.L. Mishra and Sangita Dhara
13.2 Trace Element Determinations in Marine Organisms by TXRF 206
13.3 Trace Element Determination in Blood Samples by TXRF 208
13.4 Analysis of Saliva and Oral Fluids by TXRF 209
13.5 TXRF Analysis of Hair Samples for Detection of Metal Poisoning and Other Forensic
Applications 211
13.6 Kidney Stone Analysis by TXRF 213
13.7 Elemental Analysis of Cancerous and Normal Tissues by TXRF 213
13.8 TXRF Studies on Blood and Heart Tissues as Biomarkers of Radiation Dose 214
13.9 Urine Analysis by TXRF 215
13.10 Nail Analysis by TXRF 216
13.11 Analysis of Human Eye Lens and Aqueous Humor of Cataract Patients 216
13.12 Future Prospects for TXRF Analysis of Biological Samples 217
References 217
14 Recent Developments in X-Ray Fluorescence for Characterization of Nano-Structured

Materials 219
M.K. Tiwari
14.1 Principles of GIXRF Analysis 219
14.1.1 Methodology 220
14.1.2 Phenomenon of Reflection and Refraction inside a Thin Film Medium 220
Contents xi
14.1.3 Calculation of Electric Field Intensity and Fluorescence Intensity 223

14.2 A Few Case Studies 225
14.2.1 Characterization of Ti/Co Bilayer Structures 227
14.3 Various Computational Tools (CATGIXRF Paper) 229
14.4 Structural Analysis of some Complex Nano-Structures 229
14.4.1 Trilayer Thin Film Structure 229
14.4.2 Multilayer Thin Film Structure 231
14.4.3 Analysis of Nanoparticles 235
14.4.4 Determination of Size and Shape of the Nanoparticles 236
14.5 Characterization of Absorbed Impurities on Surfaces 238
14.5.1 Introduction to Float Glass 238
14.5.2 Problem of Tin Diffusion 240
14.5.3 Experimental Measurements 241
14.5.4 GIXRF Analysis 241
14.5.5 X-Ray Reflectivity (XRR) Measurements 243
14.6 Discussion and Summary 244
References 245
15 Total-Reflection X-Ray Fluorescence Analysis of Alcoholic and Non-Alcoholic

Beverages 249
Artem S. Maltsev, Rafail A. Yusupov, and Sait A. Bakhteev
15.2 Features of Sample Preparation 253
15.2.1 Direct Analysis 253
15.2.2 Acid Digestion 254
15.3 Thin Layer Criterion 255
15.4 Quantitative Analysis 256
15.5 Angular Scanning 258
15.6 Absorption Effects 259
15.7 Method of Standard Addition 261
References 262
16 Trace Elements Analysis of Blood Samples and Serum Using Total Reflection X-Ray
Fluorescence 265
Tsenddavaa Amartaivan and Purev Zuzaan
16.2 Experimental 266
16.3 Sample Preparation 266
16.4 Applications 267
16.5 Conclusions 267
References 268
xii Contents
Part IV Beginner’s Guide 271
17 Basics and Fundamentals of X-Rays 273

Navgeet Kaur, Anju Goyal, and Rakesh K. Sindhu
17.2 Different X-Ray Excitation Sources 274
17.3 X-Ray Detectors 274
17.4 X-Ray Absorption and Scattering 275
17.5 Quantization and Detection Limits of X-Ray Fluorescence 275
17.6 Preventive Measures 276
References 276
18 General Principle, Procedures and Detectors of X-Ray Fluorescence 279

Rakesh K. Sindhu, Shantanu K. Yadav, Mansi Chitkara, Inderjeet S. Sandhu, Sandeep Arora,
Inderjeet Verma, Evren Algın Yapar, and Vivek Kumar Singh
18.2 Basic Principle of X-Ray Fluorescence 279
18.2.1 Production of X-Rays 279
18.2.2 Interaction of X-Rays with Matter 280
18.3 Small Spot Instruments and Micro-XRF 280
18.3.1 EDXRF Spectrometers with 2D Optics 281
18.3.2 EDXRF Spectrometers with 3D Optics 281
18.4 Different X-Ray Optics Configurations for Elemental Imaging in 2D/3D Using
μ-XRF 281
18.5 Conclusion 283
References 283
19 Quantitative Analysis in X-Ray Fluorescence System 287

Neslihan Ekinci, F.I. El-Agawany, and Esra Kavaz
19.2 Components for the X-Ray Spectrometry 288
19.3 Analytical Methods in X-Ray Fluorescence 290
19.3.1 The Standard Addition and Dilution Methods 291
19.3.2 Thin Film Methods 291
19.3.3 Matrix-Dilution Methods 291
19.3.4 Calibration Standardization 291
19.3.5 Internal Standardization 292
19.3.6 Standardization with Scattered X-Rays 292
19.3.7 Experimental Correction 292
19.3.8 Mathematical Correction 292
References 294
Contents xiii
20 Electronics and Instrumentation for X-Ray Fluorescence 295

Marco Carminati and Carlo Fiorini
20.2 X-Ray Sources 298
20.3 Solid-State Detectors 300
20.4 Silicon Drift Detector 302
20.5 Noise and Readout Electronics 304
20.6 Signal Processing 306
20.7 Combination with Other Techniques 306
References 307
Part V Application to Biological Samples 309
21 Energy Dispersive X-Ray Fluorescence Analysis of Biological Materials 311

Marijan Nečemer, Peter Kump, and Katarina Vogel-Mikuš
21.2 Theoretical Basics of EDXRF 312
21.2.1 X-Ray Radiation 312
21.2.2 Interaction of X-Rays with Matter 312
21.3 EDXRF Instrumentation 315
21.4 Quantification of EDXRF Spectra 317
21.5 Sampling and Sample Preparation 317
21.6 Case Studies 319
21.6.1 Elemental Profiling for Ionomic Studies 319
21.6.2 Food Authenticity Studies 320
References 323
22 X-Ray Fluorescence Analysis of Milk and Dairy Products 327

Galina V. Pashkova and Artem S. Maltsev
22.2 Conventional XRF 327
22.3 Total-reflection X-Ray Fluorescence 333
References 339
23 X-Ray Fluorescence Analysis of Medicinal Plants 341

E.V. Chuparina and А.G. Revenko
23.2 Issues Highlighted in Publications 343
23.3 XRF Specifications Used in Analysis of Medicinal Plants and Medicines 345
xiv Contents
23.4 rocedures of Plant Sample Preparation 348

P
23.5 Interelement Effects, Account Ways 349
23.6 WDXRF Analysis of Siberian Violets 352
References 356
24 X-Ray Fluorescence Studies of Animal and Human Cell Biology 363

Neera Yadav, Shilpa Chakrabarti, and Vivek Kumar Singh
24.2 Applications of XRF in Cell Biology 364
24.2.1 Measurement of Trace Elements, Contaminants and Toxins 365
24.2.2 Cellular Imaging and Measurement of Biomolecules 366
24.3 Conclusion 368
References 368
25 Toxic and Essential Elemental Studies of Human Organs Using X-Ray Fluorescence 371
Kamya Goyal, Navgeet Kaur, Anju Goyal, Rakesh K. Sindhu, and Rajwinder Kaur
25.2 Intracellular Trace Elements 374
25.2.1 Lead 374
25.2.2 Cadmium 374
25.2.3 Mercury 375
25.2.4 Iron 375
25.2.5 Iodine 375
25.2.6 Platinum 376
25.2.7 Gold 376
25.2.8 Zinc 376
25.2.9 Arsenic 376
25.3 Major Elements 376
25.3.1 Calcium 377
25.3.2 Potassium 377
25.3.3 Sodium 378
25.3.4 Magnesium 378
25.3.5 Sulfur 378
25.4 Biological Molecules 379
25.5 Non-Alcoholic and Alcoholic Beverages (Water, Tea, Must, Coffee and Wine) 380
25.6 Vegetable and Aromatic Oils 382
25.7 Conclusion 382
References 383
26 X-Ray Fluorescence for Rapid Detection of Uranium in Blood Extracted from

Wounds 387
Hiroshi Yoshii and Yukie Izumoto
Contents xv
26.2 hysical Properties of Uranium 387

P
26.3 Health Effects of Uranium Uptake 388
26.4 Current Uranium Contamination Inspection Methods 389
26.5 Usefulness of XRF Analysis in Uranium Determination 390
26.6 Examination of Blood Collection Materials 391
26.7 XRF Analysis of Simulated Uranium-Contaminated
Blood Collection Samples 392
26.7.2 XRF Device and Measurement Conditions 393
26.7.3 Results of the XRF Measurements 394
26.7.4 Peak Fitting 397
26.7.5 Calibration Curve and Detection Limit 398
26.8 Summary 401
References 401
27 X-Ray Fluorescence Analysis of Human Hair 405

Damdinsuren Bolortuya and Purev Zuzaan
27.2 Human Hair 406
27.3 Methods and Materials 407
27.3.1.1 Sampling 407
27.3.1.2 Washing 408
27.3.1.3 Drying 408
27.3.1.4 Grinding 409
27.3.1.5 Pelletizing and Special Preparations 409
27.3.1.6 Extraction/Dissolution 410
27.4 X-Ray Fluorescence Analysis 410
27.4.1 EDXRF 411
27.4.2 TXRF 411
27.4.3 WDXRF 412
27.5 Correlation of Trace Elements in Hair 412
27.6 Conclusion 413
References 413
28 X-Ray Fluorescence Spectrometry to Study Gallstones, Kidney Stones, Hair, Nails,

Bones, Teeth and Cancerous Tissues 419
Vivek Kumar Singh, M. Sudarshan, Neha Sharma, Brijbir S. Jaswal, and Onkar N. Verma
28.1 Introduction to Trace Mineral Elements in Biomedical Samples 419
28.2 Applications of XRF for Biological Specimens 420
28.2.1 XRF Applications to Calcified Tissues (Teeth and Bones) 420
28.2.2 XRF Applications for Rapid Analysis of Metallic Restorations 422
28.2.3 XRF Applications for Gallbladder and Kidney Stones Formed in Human Body 423
xvi Contents
28.2.4 XRF Applications to Blood Samples for Trace Detection 428

28.2.5 XRF Applications to Healthy and Cancerous Tissue Samples 429
28.2.6 XRF Applications to Soft Tissues and Pathological Specimens (Urine, Hair, and
Nails) 431
28.2.7 SRXRF Application to Biological Samples 434
Acknowledgement 436
References 436
29 Sampling and Sample Preparation for Chemical Analysis of Plants by Wavelength

Dispersive X-Ray Fluorescence 443
Mónica Orduña Cordero and Mª Fernanda Gazulla Barreda
29.2 Sampling and Sample Preparation 445
29.2.1 Sampling 445
29.2.2 Preparation of Plant Samples for Analysis 447
29.3 Method of Analysis: Wavelength Dispersion X-Ray Fluorescence (WDXRF)
Spectrometry 448
29.3.1 Sample Preparation for WDXRF Measurement 449
29.3.1.1 Matrix Effects 449
29.3.1.2 Mineralogical Structure and Bonding Effects 449
29.3.1.3 Particle Size 450
29.3.1.4 Preparation of Pellets 451
29.3.1.5 Preparation of Fused Beads 451
29.3.2 Wavelength-Dispersive X-Ray Fluorescence 452
29.3.2.1 Sources (X-Ray Tubes) 453
29.3.2.2 Collimators and Masks 453
29.3.2.3 Dispersive Elements 454
29.3.3 WDXRF Analysis 455
29.3.3.1 Selection and Optimization of the Instrumental Conditions 455
29.3.3.2 Calibration 457
29.3.3.3 Reference Materials 459
29.3.4 Validation of the Methodology 460
29.3.4.1 Validation Using Reference Materials 460
29.3.4.2 Validation Using Independent Methods 463
References 464
30 X-Ray Fluorescence Analysis in Medical Biology 467

Harinderjit Singh and Rakesh K. Sindhu
30.2 Role of XRF in Cancerous Diagnosis 468
30.2.1 Metals and Metalloids in Biological Systems 468
30.2.2 X-Ray Fluorescence Imaging 468
30.2.2.1 XRF Imaging of Toxic Elements 469
Contents xvii
30.2.2.2 XRF Imaging of Metals for Various Diseases 470

30.2.2.3 Pharmacology of Cobalt in Medicinal Biology 470
30.3 Conclusion and Future Prospects of XRF in Medical Biology 471
References 472
31 X-Ray Fluorescence Analysis in Pharmacology 475

Аnatoly G. Revenko
31.2 Equipment Used and Procedures for Preparation of Samples for Analysis 476
31.3 Examples of Applications of XRF for Pharmaceutical Products Research 477
31.4 Conclusion 482
References 482
Part VI Special Topics and Comparision with Other Methods 489
32 X-Ray Fluorescence and State-of-the-Art Related Techniques

to the Study of Teeth, Calculus and Oral Tissues 491
Héctor Jorge Sánchez, and Miriam Grenón
32.2 Conventional X-Ray Fluorescence Analysis 492
32.3 Synchrotron Radiation Induced XRF Analysis 494
32.4 Spatially-Resolved XRF for Studies of Bonds between Tooth and Dental Calculus 495
32.5 Total Reflection of X-Ray Fluorescence (TXRF) for Analysis of Metals in Oral Fluids of
Patients with Dental Implants 499
32.6 EDIXS Microanalysis of the Local Structure of Calcium in Tooth Layers 502
References 505
33 Lab-scale Wavelength Dispersive X-Ray Fluorescence Spectrometer and Signal

Processing Evaluation 509
Harpreet Singh Kainth, Tejbir Singh, Gurjeet Singh, Devinder Mehta, and Sanjiv Puri
33.1.1 Photon-Atom Interaction Processes 509
33.1.2 Atomic Inner-Shell Photoionization 510
33.1.3 Inner-Shell Vacancy Decay Processes 512
33.1.3.1 Radiative Transitions 512
33.1.3.2 Non-Radiative Transitions 512
33.1.4 Physical Parameters Related to Inner-Shell Vacancy Decay 513
33.1.4.1 Near-Edge Processes Contributing to Absorption of Incident Photons 513
33.1.4.2 Single Scattering 515
33.1.4.3 Multiple Scattering 515
33.1.5 Scattering Processes 516
33.1.5.1 Elastic Scattering 516
33.1.5.2 Form Factor Formalism 516
xviii Contents
33.1.5.3 Inelastic Scattering 517

33.2 Fundamental and Layout 519
33.2.1 Experimental Techniques for Investigation of the Photon-Atom Interaction
Processes 519
33.2.2 Photon Sources 520
33.2.3 Specimen Target 521
33.2.4 Radiation Detectors 521
33.2.5 WDXRF Spectrometer 522
33.2.6 Target Preparation 524
33.2.7 Detection System 525
33.2.8 Intensity Correction Method 526
33.2.9 Energy Resolution and Efficiency 528
33.3 Qualitative and Quantitative Analysis 529
33.3.1 Sample Preparation for Calibration Curves 533
33.3.2 Sensitivity of WDXRF Instrument 533
33.3.3 Instrumental Limit of Detection 534
33.4 Applications 534
33.4.1 Chemical Effects and Speciation in k or l X-Ray Emission Lines 534
33.5 Conclusion and Prospects 543
Acknowledgment 545
References 545
34 Chemometric Processing of X-Ray Fluorescence Data 551

Vitaly Panchuk, Valentin Semenov, and Dmitry Kirsanov
34.2 Principal Component Analysis 553
34.3 Hierarchical Cluster Analysis 556
34.4 Partial Least Squares (PLS) 557
34.5 Other Methods 561
References 561
35 X-Ray Crystallography in Medicinal Biology 563

Shilpa Chakrabarti and Neera Yadav
35.2 Drug Design 563
35.2.1 XRC in Antiparasitic Drugs 563
35.2.2 XRC and XRF in Anti-Cancer and Anti-Diabetic Drugs 564
35.3 Monitoring Changes in Concentrations of Trace Elements 564
35.3.1 XRF and Autoimmune Diseases 564
35.3.2 XRC and XRF in Cardiac Function 564
35.3.3 XRC in Detection of Bone Loss 565
35.3.4 XRF in Elemental Analysis in Implants 565
35.3.5 XRF in Study of Pathological Specimens 565
Contents xix
35.3.6 XRF Use in Recognizing Dental Caries 565

35.3.7 XRF in Detection of Trace Elements 566
35.4 Conclusion 566
References 566
36 Historical Fundamentals of X-Ray Instruments and Present Trends in Biological

Science 569
Kanishka Rawat, Neha Sharma, and Vivek Kumar Singh
36.1 Brief History of X-Ray Fluorescence 569
36.3 Nature of X-Rays 572
36.3.1 Properties of X-Rays 573
36.3.2 Hard and Soft X-Rays 573
36.3.3 Continuous Spectrum 574
36.3.4 Characteristic X-Ray Spectrum 574
36.4 Production of X-Rays 575
36.4.1 Production by Electrons 575
36.4.2 Production in Lightning and Laboratory Discharges 575
36.4.3 Production by Fast Positive Ions 575
36.5 Interaction of X-Rays with Matter 576
36.5.1 X-Ray Absorption and Scattering 576
36.6 Role of X-Rays in Biological Analysis 576
36.7 Different X-Ray Excitation Sources 580
36.8 X-Ray Detectors 583
36.8.1 Photographic Film 583
36.8.2 Semi-Conductor Detectors 584
36.8.3 Gas-Filled Detectors 584
36.9 Polarization of X-Rays 585
36.10 Quantization and Detection Limits of X-Rays 586
36.11 Preventative Measures 586
References 587
37 X-Ray Fluorescence Studies of Biological Objects in Mongolia 591

P. Zuzaan and D. Bolortuya
37.2 Determination of Some Elements in Plant Materials of the Khuvsgul Lake Basin 591
37.2.1 Preparation of Plant Samples 592
37.2.2 Sample Preparation for Measurement 593
37.2.3 Measurements and Methods 593
37.2.4 Procedure of Analysis 594
37.3 Human Hair Studies in Mongolia 597
37.3.1 The Human Hair Study for Medicine of Mongolia 597
xx Contents
37.3.2 Distribution of Calcium Content in Mongolians’ Hair 599

37.4 Application of X-Ray Fluorescence Analysis for Forensic Investigations in
Mongolia 600
37.5 Determination of Some Trace Elements in Livestock Using XRF 601
37.6 Determination of Some Trace Elements in Foods Using XRF 602
References 604
38 Arsenic Analysis 609

Jun Kawai
38.2 Arsenic Species 610
38.3 Gutzeit Method 611
38.4 Principles of HG-AAS Arsenic Analysis 611
38.5 Problems in Yamauchi’s Method 613
38.5.1 Glass Test Tube 613
38.5.2 NaOH Decomposition 614
38.5.3 pH Values for Speciation 615
38.5.4 Detection Limit 615
38.6 Selective Excitation of SRXRF 615
38.7 Stray Light 616
References 619
39 X-Ray Fluorescence: Current Trends and Future Scope 623

Rakesh K. Sindhu, Shantanu K. Yadav, Arashmeet Kaur, Manish Kumar, and Pradeep Kumar
39.2 Principle 624
39.3 X-Ray Fluorescence 625
39.3.1 Microanalysis 625
39.3.2 Particles Dispersive X-Ray Spectroscopy 625
39.3.3 The Behavior of X-Rays 625
39.3.4 X-Ray Intensity 625
39.3.5 Process 626
39.3.6 Synchrotron XRF (SR-XRF) 626
39.4 Application of X-Ray Fluorescence Technique 626
39.4.1 Pharmacological Action 626
39.4.2 XRF Soft Tissue and Pathological Samples Application 627
39.4.3 In Tooth Analysis 628
39.5 XRF Technique Used in Biology 628
39.5.1 Detection of Metal Ion(s) 628
39.5.1.1 Role of Metals in Biology 628
Contents xxi
39.6 pplications of XRF in the Study of Plant Physiology 629

A
39.6.1 Hyperaccumulating Plant 629
39.6.2 Accumulators and Hyper-Sensors of Selenium 630
39.6.3 Accumulators and Hyperaccumulators of Arsenic 630
39.6.4 Accumulators and Hyperaccumulators of Cadmium 631
39.7 Application in Animal Biology and Medicinal Biology 632
39.7.1 Application in Health Science 632
39.7.1.1 Mercury Toxicology 632
39.7.1.2 Arsenic Toxicology 634
39.7.1.3 Iatrogenic Toxic Metals 635
39.7.1.4 Neurodegenerative Ailments 636
39.8 Applications in Nanotechnology 637
39.8.1 Potential Therapeutics and Xenobiotic Labels 637
39.9 Methodological Improvement 637
39.9.1 Magnetic Resonance Imaging 637
39.9.2 Mass Spectrometry Imaging 637
39.10 Molecular Fluorescence Samples 638
39.10.1 Mercury 639
39.10.2 Copper 639
39.10.3 Zinc 639
39.11 Fourier Transform Infra-red (FTIR) Spectroscopy 640
39.12 Novel X-Ray Imaging Methods 641
39.13 Conclusion and Advances 642
References 642
Index 647
xxiii
List of Contributors
Tsenddavaa Amartaivan Sandeep Arora

Department of Physics Chitkara College of Pharmacy
School of Arts and Sciences Chitkara University
National University of Mongolia Punjab
Ulaanbaatar India
Mongolia
Volkan Aylikci
and Center for Science and Technology Studies and
Nuclear Research Center Research
National University of Mongolia Iskenderun Technical University-ISTE
Ulaanbaatar Hatay
Mongolia Turkey
and
Evren Algın Yapar
Analysis and Control Laboratories Department Department of Metallurgical and Material
Turkish Medicines and Medical Science Engineering
Devices Agency Iskenderun Technical University–ISTE
MoH Hatay
Ankara Turkey
Turkey
Ersin Bahceci
Center for Science and Technology Studies and
Iztok Arcŏn
Research
Department for Low and Medium
Iskenderun Technical University-ISTE
Energy Physics
Hatay
Jožef Stefan Institute
Turkey
Ljubljana
Slovenia and
and Department of Metallurgical and Material

Science Engineering
Laboratory of Quantum Optics
Iskenderun Technical University–ISTE
University of Nova Gorica
Hatay
Nova Gorica
Turkey
Slovenia
xxiv List of Contributors
Sait A. Bakhteev Tolga Depci

Department of Analytical Chemistry Department of Petroleum and Natural Gas
Certification and Quality Management Engineering
Kazan National Research Technological Iskenderun Technical University–ISTE
University Hatay
Kazan Turkey
Russia
Sangita Dhara
Damdinsuren Bolortuya Fuel Chemistry Division
Nuclear Research Center Bhabha Atomic Research Centre
National University of Mongolia Mumbai
Ulaanbaatar India
Mongolia
and
Marco Carminati Homi Bhabha National Institute
Dipartimento di Elettronica Mumbai
Informazione e Bioingegneria India
Politecnico di Milano
Milano F.I. El-Agawany
Italy Department of Physics
Faculty of Science
and Menoufia University
Istituto Nazionale di Fisica Nucleare Shebin El-Koom
(INFN), Milano Menoufia
Milano Egypt
Italy
Neslihan Ekinci
Department of Physics
Shilpa Chakrabarti
Faculty of Science
Biochemistry Department
Ataturk University
University of Allahabad
Erzurum
Prayagraj
Turkey
India
Mª Fernanda Gazulla Barreda
Mansi Chitkara Institute of Ceramic Technology-Ceramic
Chitkara University Industries Research Association
Institute of Engineering and Technology Jaume I. Castellón University
Chitkara University Castellón de la Plana
Punjab Spain
India
Carlo Fiorini
E.V. Chuparina Dipartimento di Elettronica
Vinogradov Institute of Geochemistry Informazione e Bioingegneria
SB RAS Politecnico di Milano
Irkutsk Milano
Russian Federation Italy
List of Contributors xxv
and Héctor Jorge Sánchez

Faculty of Mathematics, Physics, Astronomy,
Istituto Nazionale di Fisica Nucleare
and Computer Science
(INFN), Milano
National University of Córdoba
Milano
Córdoba
Italy
Argentina
Anju Goyal and
Chitkara College of Pharmacy
Instituto de Física Enrique Gaviola
Chitkara University
CONICET
Punjab
Córdoba
India
Argentina
Kamya Goyal
Abdelhalim Kahoul
Laureate Institute of Pharmacy
Department of Materials Science
Kathog, Jawalamukhi, Kangra
Faculty of Sciences and Technology
Himachal Pradesh
Mohamed El Bachir El Ibrahimi University
India
Bordj-Bou-Arreridj
and Algeria
Chitkara College of Pharmacy and
Chitkara University
Laboratory of Materials Physics
Patiala
Radiation and Nanostructures (LPMRN)
Punjab
Mohamed El Bachir El Ibrahimi University
India
Bordj-Bou-Arreridj
Algeria
Miriam Grenón
Faculty of Dentistry
National University of Córdoba Navgeet Kaur
Córdoba Chitkara College of Pharmacy
Argentina Chitkara University
Punjab
Yukie Izumoto India
National Institute of Radiological Science and
National Institutes for Quantum Science and
Technology Swami Devi Dyal Institute of Pharmacy
Inage-ku Panchkula
Chiba Haryana
Japan India
Brijbir S. Jaswal Rajwinder Kaur

Department of Physics Chitkara College of Pharmacy
University of Lucknow Chitkara University
Lucknow Patiala
Uttar Pradesh Punjab
India India
xxvi List of Contributors
Arashmeet Kaur Pradeep Kumar

Chikara College of Pharmacy Department of Pharmaceutical Sciences and
Chikara University Natural Products
Punjab Central University of Punjab
India Bathinda
India
Esra Kavaz
Department of Physics Vivek Kumar Singh
Faculty of Science Department of Physics
Ataturk University University of Lucknow
Erzurum Lucknow
Turkey Uttar Pradesh
India
Jun Kawai
Department of Materials Science and Peter Kump
Engineering Department for Low and Medium
Kyoto University Energy Physics
Sakyo-ku Jožef Stefan Institute
Kyoto Ljubljana
Japan Slovenia
Nuray Kup Aylikci

Dmitry Kirsanov
Department of Engineering Sciences
Institute of Chemistry
Iskenderun Technical University–ISTE
St. Petersburg State University
Hatay
Saint Petersburg
Turkey
Russia
and
Alojz Kodre Institute of Graduate Studies
Department for Low and Medium Department of Energy Systems
Energy Physics Engineering
Jožef Stefan Institute Iskenderun Technical University–ISTE
Ljubljana Hatay
Slovenia Turkey
and and
Faculty for Mathematics and Physics Center for Science and Technology Studies and
University of Ljubljana Research
Ljubljana Iskenderun Technical University-ISTE
Slovenia Hatay
Turkey
Manish Kumar
Chikara College of Pharmacy Changling Lao
Chikara University Guilin University of Technology
Punjab Guilin
India China
List of Contributors xxvii
and Devinder Mehta

National Research Center of Geoanalysis
Panjab University
Beijing
Chandigarh
China
India
and
N.L. Mishra
China University of Geosciences (Beijing)
Fuel Chemistry Division
Beijing
Bhabha Atomic Research Centre
China
Mumbai
India
Jian Liu
College of Environmental Natural and
Resource Sciences
Homi Bhabha National Institute
Zhejiang Provincial Key Laboratory of
Mumbai
Agricultural Resources and Environment
India
Zhejiang University
Hangzhou Yeasmin Nahar Jolly
China Atmospheric and Environmental Chemistry
Liqiang Luo Laboratory
National Research Center of Geoanalysis Chemistry Division, Atomic Energy Centre
Beijing Bangladesh Atomic Energy Commission
China Dhaka
Bangladesh
Artem S. Maltsev
Center for Geodynamycs and Geochronology Marijan Nečemer
Institute of the Earth’s Crust Department of Low and Medium
SB RAS Energy Physics
Irkutsk Jožef Stefan Institute
Russia Ljubljana
Slovenia
and
Department of Analytical Chemistry Mónica Orduña Cordero
Certification and Quality Management Institute of Ceramic Technology-Ceramic
Kazan National Research Technological Industries Research Association
University Jaume I. Castellón University
Kazan Castellón de la Plana
Russia Spain
Eva Marguí Onder Oruc

Department of Chemistry Institute of Graduate Studies
University of Girona Department of Energy Systems Engineering
Girona Iskenderun Technical University–ISTE
Spain Hatay
Turkey
xxviii List of Contributors
Vitaly Panchuk Anatoly G. Revenko

Institute of Chemistry Institute of the Earth’s Crust
St. Petersburg State University SB RAS
Saint Petersburg Irkutsk
Russia Russian Federation
Galina V. Pashkova Inderjeet S. Sandhu

Institute of the Earth’s Crust Chitkara University
SB RAS Institute of Engineering and Technology
Irkutsk Chitkara University
Russia Punjab
India
Paula Pongrac
Department of Biology Valentin Semenov
Biotechnical Faculty Institute of Chemistry
University of Ljubljana St. Petersburg State University
Ljubljana Saint Petersburg
Slovenia Russia
and
Neha Sharma
Department for Low and Medium
Energy Physics
University of Lucknow
Jožef Stefan Institute
Lucknow
Ljubljana
Uttar Pradesh
Slovenia
India
Sanjiv Puri and
Department of Basic and Applied Sciences
Punjabi University School of Physics
Patiala Shri Mata Vaishno Devi University
India Katra
Jammu and Kashmir
Ignasi Queralt India
Institute of Environmental Assessment and
Water Research
Darya S. Sharykina
IDAEA-CSIC
Institute of the Earth’s Crust
Barcelona
SB RAS
Spain
Irkutsk
Kanishka Rawat Russian Federation
Applied Nuclear Physics Division
Saha Institute of Nuclear Physics
Yating Shen
Kolkata
National Research Center of Geoanalysis
West Bengal
Beijing
India
China
List of Contributors xxix
Rakesh K. Sindhu M.K. Tiwari

Chitkara College of Pharmacy Synchrotrons Utilization Section
Chitkara University Raja Ramanna Centre for Advanced
Rajpura Technology (RRCAT)
Punjab Indore
India India
and
V.A. Trunova
Swami Devi Dyal Institute of Pharmacy Nikolaev Institute of Inorganic
Panchkula Chemistry SB RAS
Haryana Novosibirsk
India Russia
Harinderjit Singh Onkar N. Verma

Adesh Institute of Pharmacy & Biomedical Department of Physics
Sciences University of Lucknow
Adesh University Lucknow
Bathinda Uttar Pradesh
Punjab India
India
Inderjeet Verma
Harpreet Singh Kainth M.M. College of Pharmacy
Department of Basic and Applied Sciences MM(DU)
Punjabi University Mullana-Ambala
Patiala India
India
Katarina Vogel-Mikuš
Tejbir Singh Department of Low and Medium
Department of CIL/SAIF Energy Physics
Panjab University Jožef Stefan Institute
Chandigarh Ljubljana
India Slovenia
and
Gurjeet Singh
Department of Biology
Biotechnical Faculty
Punjabi University
University of Ljubljana
Patiala
Ljubljana
India
Slovenia
M. Sudarshan
Neera Yadav
UGC-DAE Consortium for Scientific Research
College of Pharmacy
Kolkata
Gachon University of Medicine and Science
India
Incheon City
South Korea
xxx List of Contributors
Shantanu K. Yadav Rafail A. Yusupov

Chikara College of Pharmacy Department of Analytical Chemistry
Chikara University Certification and Quality Management
Punjab Kazan National Research Technological
India University
Kazan
Hiroshi Yoshii Russia
National Institute of Radiological Science
National Institutes for Quantum Science and Shuai Zhu
Technology National Research Center of Geoanalysis
Chiba Beijing
Japan China
Jing Yuan Purev Zuzaan

East China Center for Geoscience Innovation Nuclear Research Center
Nanjing Center of Geological Survey National University of Mongolia
China Geological Survey Ulaanbaatar
Nanjing Mongolia
China
xxxi
Preface
X-ray fluorescence (XRF) spectroscopy is a well-established analytical technique being used extensively
for mining, metallurgy, petroleum, and geological studies, though not widely used for biological
applications. During the past decade, XRF spectrometry has gone through major changes in the field of
biological sciences. This book is a guide which provides an up-to-date review of XRF spectrometry for
biological, medical, food, environmental, and plant science researchers. It covers the basic principles
and latest developments in instrumentation and applications of X-ray fluorescence in biological
sciences. This also provides a thoroughly updated and expanded overview to industry professionals in
X-ray analysis over the last decades. The main feature of this book is that it provides information about
XRF techniques and procedures for qualitative and quantitative analysis of biological specimens worth
modern applications and industrial trends.
The chapters are contributed by independent groups in the world. Four chapters are contributed
by the members of editorial advisory board of the journal “X-Ray Spectrometry” from Wiley. The
chapters are divided into six parts. Part 1 is a general introduction of XRF. Parts 2 and 3 are most
advanced methods of SR-XRF and TXRF, which are micro-XRF, high sensitivity (low detection
limit) XRF. Part 4, a beginner’s guide, is one of the characteristics of the present book. Parts 5 and
6 are the main parts of the present book.
Part I (General Introduction) consists of seven (07) chapters. Chapter 1 describes about the XRF
and comparison with other analytical methods such as AAS, ICP-AES, LA-ICP-MS, IC, LIBS,
SEM-EDS, and XRD. Chapter 2 highlights the significant role of different XRF configurations for
both multi-elemental bulk analysis and element distribution within vegetal tissues. In Chapter 3,
the application of XRF analysis is described for the chemical compositions of tea and coffee sam-
ples. Chapter 4 deals with total reflection X-ray fluorescence (TXRF) spectrometry and its suitabil-
ity for biological samples. In this chapter, the fundamentals, basic principles, and theoretical
aspects of TXRF have been discussed along with its advantages and limitations. Chapter 5 describes
the use and application of μ-XRF and XANES to understand the interaction process and mecha-
nism between microorganisms and heavy metal. Chapter 6 covers the details of EDXRF techniques
for the application to clinical samples such as blood and hair. In Chapter 7, all considerations
related with the sample preparation process are summarized which is very crucial for XRF analysis.
Part II (Synchrotron Radiation XRF) consists of five (05) chapters which show how Synchrotron
Radiation XRF (SRXRF) can be used to provide analytical information in biological sciences for
elemental composition. Chapter 8 covers numerous aspects of SRXRF and its applications. This
chapter highlights the usefulness of XRF technique for the elemental characterization of different
sample matrixes in non-destructive manners. Chapter 9 deals with the application of SR-based
micro-XRF spectroscopy for plants. Chapter 10 covers the application of μ-XRF to study toxic
elements in plants. Chapter 11 highlights the application of micro-XRF for the analysis of
xxxii Preface
benthonic fauna (earthworm and nematodes) in soils and sediments. Chapter 12 discusses in
detail the use of SRXRF for the analysis of microelements in biopsy tissues.
Part III (Total Reflection XRF) consists of four (04) chapters which describe in detail the princi-
ples and basic fundamentals of total reflection XRF (TXRF) along with their biological applications.
Chapter 13 covers the applications of TXRF for the trace element determinations in marine
organisms, blood samples, saliva and oral fluids, hairs, nails, kidney stones, urine samples, and
forensic samples. Chapter 14 demonstrates the applications of combined X-ray reflectivity (XRR)
and grazing incidence X-ray fluorescence (GIXRF) technique for the characterization of thin films
and nano-structured materials. Chapter 15 deals with the analysis of alcoholic and non-alcoholic
beverages by TXRF. Chapter 16 describes the details of using TXRF and XRT techniques for trace
elemental analysis of blood and serum samples.
Part IV (Beginner’s Guide) consists of four (04) chapters which cover the basics theory of XRF and
historical fundamentals of XRF instruments, quantitative analysis methods, electronics and instru-
mentation, methods of using XRF to study biological samples. Chapter 17 introduces the atomic
physics of the XRF spectrometry which is very useful for beginners to learn for its applications.
Chapter 18 includes general principles, production, and detectors of X-ray waves. Chapter 19
introduces general discussion on quantitative analysis methods and procedures which is the princi-
pal subject of XRF spectroscopy. Chapter 20 deals with the crucial aspects concerning the operation
and optimization of electronics for X-ray detection and fluorescence spectrometry.
Part V (Application to Biological Samples) consists of eleven (11) chapters which include the dif-
ferent biological applications of XRF spectrometry. Chapter 21 highlights the theoretical basics of
the EDXRF followed by some relevant case studies such as elemental profiling for ionomic studies
and food authenticity studies. Chapter 22 deals the application of XRF including TXRF to milk and
dairy products. Chapter 23 includes the literature review on the elemental concentration analysis of
medical plants using XRF technique. Chapter 24 deals with the application of XRF in animal and
human cell biology. Chapter 25 covers a variety of biomedical applications using XRF spectrometry.
Chapter 26 describes the usefulness of XRF technique to analyze uranium (U) in blood extracted
from wounds. Chapter 27 highlights the use of XRF for the analysis of human hair. Chapter 28
discusses the potential utility of XRF methods to analyze different kinds of biological samples such
as calcified/dental tissues, gallbladder and kidney stones, hair, nails, blood, urine, and clinical
samples. Chapter 29 describes the principles of using WDXRF for the chemical analysis of plant
samples. Chapter 30 covers the use and application of XRF in medicinal biology. Chapter 31
describes in details the use of XRF in pharmacology.
Part VI (Special Topics and Comparison with Other Methods) consists of eight (08) chapters.
This section includes some chapter based on special topics and comparisons of XRF with other tech-
niques. Chapter 32 describes the XRF technique and state-of-the-art related techniques specifically
as they regard the study of teeth, tartar, and oral tissues. Chapter 33 describes in details the princi-
ples, theory and applications of WDXRF spectrometry. Chapter 34 describes the chemometric pro-
cessing of XRF data which is one of the most important steps in XRF spectrometry. Chapter 35
briefly describes the applications of X-ray crystallography in medicinal biology. Chapter 36
describes the historical fundamentals of X-ray instruments and present trends in the field of bio-
logical science. Chapter 37, the application and development of XRF spectrometry is discussed for
biological objects in Mongolia. Chapter 38 highlights the developments and use of XRF techniques
to study arsenic in biological samples in Japan. Chapter 39 is the most important chapter which
describes about the current trends and future prospects of XRF technique.
Four chapters (17, 18, 25, and 30) are at very basic level, which will be useful for biologists to
understand XRF and its biological applications. The priority is understandable by trading-off the
accuracy or precise expression in these four chapters.
Preface xxxiii
We would like to take this opportunity to express our gratitude to all of the authors for their
excellent contributions in this book. We hope the readers will enjoy this book “X-Ray Fluorescence
in Biological Sciences: Principles, Instrumentation and Applications” and that it contributes to the
continued instrumental developments of XRF and biological applications. We also hope that it
encourages and inspires the beginners to the field in exploring the multifaceted aspects of XRF.
Finally, the critical evaluations and recommendations by the reviewers for the applicability of
the XRF methods to biological samples will make this book a valuable asset for anyone employing
or improving upon these techniques.
December, 2021 Dr. Vivek Kumar Singh

Professor Jun Kawai
Dr. Durgesh Kumar Tripathi
1
Part I
General Introduction
3
X-Ray Fluorescence and Comparison with Other Analytical

Methods (AAS, ICP-AES, LA-ICP-MS, IC, LIBS, SEM-EDS, and XRD)
Kanishka Rawat1, Neha Sharma2, and Vivek Kumar Singh3
1
Applied Nuclear Physics Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India
2
School of Physics, SMVD University, Katra, 182320, Jammu and Kashmir, India
3
Department of Physics, University of Lucknow, Lucknow, 226007, Uttar Pradesh
1.1 Introduction
Most highly complex structured materials require good analytical techniques that can furnish
information about the spatially distributed elements in the materials and permit the examination
of their structures. Many analytical methods exist which provide insight into the chemical compo-
sitions and structure of the materials. Each technique has its own advantages and limitations in
terms of analytical performance, sensitivity, accuracy, and applicability. X-ray fluorescence (XRF)
is an elemental analysis technique that is used for elemental and chemical analysis of various
materials including glass, metals, and ceramics. XRF is also seeing increased application and
greater utility in the analysis of biological materials [1–3]. In XRF analysis, X-ray photons
characteristic of the elemental makeup of the sample material are emitted as it is bombarded with
highly energetic X-ray beams [1–3]. In most circumstances, XRF is considered non-destructive.
The other factors responsible for its wide adoption are low cost of sample preparation, relative
ease, and stability.
Several elemental analysis techniques such as laser induced breakdown spectroscopy (LIBS),
inductively coupled plasma mass spectroscopy (ICP-MS), ion chromatography (IC), etc. are widely
used for the analysis of materials, particularly biological samples such as tooth, bone, nail, stone,
blood, cancerous tissues, etc. [4–7]. There are many other similar methods such as time-of-flight
secondary ion mass spectrometry (TOF-SIMS) and proton-induced X-ray emission spectroscopy
(PIXE) that have many important biomedical applications. Inductively coupled plasma (ICP) is
also one of them, which is a plasma source wherein energy is supplied by electric currents gener-
ated by electromagnetic induction [3]. ICP has numerous applications such as in nuclear technolo-
gies, isotopic speciation, and detection of chemical elements. IC separates polar molecules and
ions on the basis of their chemical affinity with regards to the ion exchanger [3]. It can be operated
on all charged molecules, like bio-molecules (especially amino acids), large proteins, small nucleo-
tides etc. It has many clinical and industrial applications. In this chapter, we present briefly
the position of XRF including micro-XRF (μ-XRF) among some other the analytical methods
including ICP-AES/MS, IC, LIBS, TOF-SIMS, and PIXE.
X-Ray Fluorescence in Biological Sciences: Principles, Instrumentation and Applications, First Edition.
Edited by Vivek Kumar Singh, Jun Kawai, and Durgesh Kumar Tripathi.
© 2022 John Wiley & Sons Ltd. Published 2022 by John Wiley & Sons Ltd.
4 1 X-Ray Fluorescence and Comparison with Other Analytical Methods
1.2 Analytical Capabilities of XRF and Micro-XRF
XRF spectrometry is generally used in two different kinds of configurations: wavelength dispersive
mode (WD-XRF) and energy dispersive mode (EDXRF) [8, 9]. Both of them have different ways of
detecting and analyzing emitted fluorescent X-ray photons. ED-XRF spectrometers have detection
systems which examine the distinct energies of the X-ray photons coming directly from the sample
material. The XRF spectrum is generated by detecting and plotting the relative count numberings
of X-rays at each energy value. The energy dispersive detectors basically involve the creation of
electron–hole pairs in semiconductor materials (Si). After the emergence of silicon drift detectors
(SDD), EDXRF is mainly used. As compared with EDXRF, WDXRF is quite expensive and is not
needed for testing materials for steel industry or ceramics industry, for which EDXRF is enough. In
recent years, EDXRF leads over WDXRF and is a powerful tool for elemental analysis to determine
major, minor, and trace elements in biological samples [3]. EDXRF spectrometers are simpler in
design, smaller, and more cost effective than other technologies. Examples of some common
EDXRF applications include: quantifying atomic elements in: food, animal feed, cosmetics, woods,
toothpaste, cement, kaolin clay, granular catalysts, ores, and many others.
One more difference between the techniques is that with an EDXRF system, the full spectrum is
obtained virtually at once. So, a range of elements belonging to the periodic table can be determined
simultaneously. With an WDXRF system, the spectrum has been procured by a series of discrete step,
which is time-consuming, and also expensive due to the restricted number of detectors.
1.2.1 Micro-XRF
XRF is a bulk technique with the analysis range varying from several millimeters to several centim-
eters. Inhomogeneous samples compacted into a pellet form and thereby make it little time con-
suming. Also it requires a large amount of sample material for the analysis. Many advancements
have been made in the field of X-ray optics that gave rise to originate to narrow X-ray beams (1 mm
to 10 μm). Such developments allow even a solo microscopic particle to be discretely analyzed for
an explicit elemental image of high spatial resolution.
XRF [7] is based on an energy-dispersive detection system. For the generation of precise and
accurate elemental images consisting of thousands of pixels, a fast acquisition is needed at each
and every pixel position. When using a simple WDXRF spectrometer, the scanning procedure is
time consuming and does not support the imaging applications as provided by XRF. XRF has broad
range of research applications including geology, mineralogy, gemology, archaeology, motor
engineering, electronics, pharmaceutics, environmental studies, and biomedicine.
1.3 Comparison with Other Analytical Methods
1.3.1 Overview
Owing to advancements in XRF spectrometry, EDXRF systems are used in combination with scan-
ning electron microscopy (SEM-EDS) to determine elemental constituents at small scales.
Importantly, XRF is used with synchrotron radiation sources (SRXRF) which is very similar to μ-
XRF that covers numerous applications.
There exist many analytical techniques such as XRF, μ-XRF, SRXRF, total reflection X-ray spec-
troscopy (TXRF), atomic absorption spectrometry (AAS), laser-induced breakdown spectroscopy
1.3 Comparison with Other Analytical Method 5
(LIBS), laser ablation inductively coupled plasma mass spectroscopy (LA-ICP-MS), inductively
coupled plasma optical emission spectrometry (ICP-OES), time-of-flight secondary ion mass spec-
trometry (TOF-SIMS), PIXE, etc. which are currently being used for elemental analysis of materi-
als, including biological samples. Some important parameters that distinguish the analytical
capabilities of the techniques such as elemental range, imaging possibility, depth resolution, and
instrumental effort are summarized in Table 1.1 [3, 8–11].
All the techniques have their own advantages and different analytical capabilities that can be
used to analyze different kinds of materials. Also, the instrumental efforts of these techniques are
also different and thus, some techniques require more or less effort on the part of the operator.
PIXE and synchrotron radiation-XRF require high instrumental effort [3, 8, 9]. Additionally, com-
plex sample handling is necessary in ultrahigh vacuum for Auger electron spectrometry, transmis-
sion electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and secondary ion mass
spectrometry (SIMS) [3, 8]. On the other hand, a complex laser interaction with the samples occurs
in LA-ICP-MS. However, a few methods with a restricted spatial resolution, such as conventional
XRF or atomic emission spectroscopy (AES), are also available and are often used for elemental
analysis in comparison to above techniques.
The methods discussed above produce similar information about the sample compositions and
in most cases they provide complementary information. The utility of these techniques depends on
their analytical performance and availability particularly their costs.
Figure 1.1 shows a concise visual reference for comparing analytical techniques used for materi-
als characterization, elemental analysis, evaluation surface analysis, and purity surveys etc. in
terms of their detection limits and analytical resolutions.
1.3.2 Inductively Coupled Plasma (ICP) Analysis

ICP spectroscopy is the most popular analytical method to detect and quantify the chemical ele-
ments present in a given sample. This is based on the ionization of samples by extremely hot
plasma, usually made from argon gas [4]. In the next Sections 1.3.2.1 and 1.3.3, we have briefly
discussed the working principles and analytical capabilities of two forms of ICP spectroscopy such
as ICP-MS and ICP-AES.
1.3.2.1 Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

ICP-MS uses inductively coupled plasma to ionize the atoms [3, 4]. It is used to atomize the sample,
creating polyatomic ions, which are then detected. The ions from the plasma are separated through
a series of cones into a mass spectrometer, usually a quadrupole. The extraction of ions is based on
their mass-to-charge ratio. An ionic signal is received by a detector which is correlated to the con-
centrations of atoms in the sample. These concentrations are obtained by drawing the calibration
curves using some certified reference materials (CRMs).
It has the capability to investigate several metals as well as non-metals in the liquefied samples
at milligram to nanogram levels per liter. It is also used for isotopic analysis of the elements. This
technique has been recognized as one of the most important analytical techniques in a variety of
industries for the monitoring of impurities in semiconductor manufacturing, environmental mon-
itoring, geochemical analysis, mining and metallurgy, pharmaceutical industries, and biomedical
analysis. ICP-MS has a good precision, sensitivity, and greater speed. The main disadvantage of
ICP-MS is that it produces many interfering species like component gases of air, argon from the
plasma, and contamination from glassware and cones used.
Table 1.1 Overview of some analytical techniques including AAS, ICP, LIBS, XRF, μ-XRF, SR-XRF, and TOF-SIMS [3, 8–11].
Resolution
Excitation Instrument Scan size/area
Techniques source Elements detected Spatial Depth Detection limit Imaging efforts analysis (mm) Specific remarks
XRF X-rays B-U (WDXRF); 20 mm 10 nm 1–100 ppm for No Medium ~30 μm (EDXRF) Non-destructive
Na-U (EDXRF) most elements and ~500 μm
(WDXRF)
μ-XRF X-rays Multi-element 20–500 μm 10 nm 20–50 ppm Yes Medium Upto 190 × 160 Relatively slow, risk for radiation
(Al-U) damage
μ-SRXRF X-rays Multi-element >10 nm 1 nm 5 ppm Yes Very high
TXRF X-rays Na-U >3 nm Yes High ●● Polished surface required for
(Optional) best detection limits, Can
analyze many substrates, e.g. Si,
SiC, GaAs, InP, sapphire, glass
X-ray fluorescence X-rays Multi-element 0.05–1 μm >100 μm <0.1 ppm Yes Medium Upto 150 × 100 Ability for spectroscopy (XAS) to
microscopy (Al-U but poor determine chemical speciation
(XFM) 2nd row Z > 42
SEM/TEM-EDS Electrons Multi-element <0.5 μm <0.5 μm 1000 ppm Yes Medium 7×7 Resolution depends on element
(O-U) investigated
PIXE Protons (for Multi-element 2 μm 10–100 μm 1–10 ppm Limited Very high 4×4 Quantitative measurements of
biological (Na-U) heavier elements that can’t be
applications) resolved by RBS alone
XPS/ESCA X-rays Li-U (Chemical 0.5 nm 3 nm 100 ppm Yes High Smallest ●● Limited specific organic
bonding analytical area information and sample
information) ~10 μm compatibility with UHV
environment.
Auger (AES) Electrons Li-U 0.2 μm 3 nm 100 ppm Yes High Small area ●● Analysis of insulators can be
analysis (~20 nm difficult and samples must be
minimum) vacuum compatible.
0005270020.INDD 6 03-01-2022 17:50:15

SIMS Ions H-U including 5 μm 0.1 nm 1 ppb Yes Medium ●● Small-area ●● Destructive, no chemical
isotopes analysis bonding information, and
(1–10 μm) sample must be solid and
vacuum compatible.
TOF-SIMS Ions Full periodic <0.1 μm 1 nm 1 ppm Yes High ●● Samples must be vacuum
table coverage, compatible
plus molecular
species
RBS He2+ Ions B-U 2 mm 10–20 nm 1–1000 ppm No Medium ●● Large analysis ●● Non-destructive, Conductor and
(alpha particles) area (~2 mm) insulator analysis
ICP-OES Argon plasma Li-U (except No Bulk <1 ppb No Medium ●● No ●● C, H, N, O and halogens cannot
gases, halogens, chemical be determined.
low quantity of P analysis
and S) technique
ICP-MS Plasma Most of the No No Typically No Medium ●● No ●● Polyatomic mass interferences,
source elements ng/ L atmospherics and light halogens
LA-ICP-MS Photos (Laser) Multi-element 100 nm 0.1–1 μm <1 ppb Yes Medium ●● 20 × 20 ●● Elemental, stable isotope
(Al-U) (Limited: distribution analysis and
ablated mapping
surface)
LIBS Photos (Laser) ●● All elements >0.1 μm 1 ppm Yes Medium ●● Strong matrix-effects on
detectable (Limited) emission spectra
2
Atom Probe Laser or voltage ●● H-U <1 nm 0.3 nm ~10 ppm Yes High ●● 50 × 50 nm ●● Ability to identify isotopes,
Tomography pulse Cluster analysis for nanoscale
(APT) precipitates
STEM Electrons ●● B-U (EDS) 3 nm 3 nm Typically ppm Yes High ●● 5 μm x 5 μm ●● Strong contrast between
crystalline vs amorphous
materials without chemical
staining
AAS Radiation ●● Most of the Typically μg/l No Medium ●● No ●● Destructive, time consuming,
Sources (Hollow elements sequential analysis of 1 to 6
Cathode Lamps except some elements, separate method of
HCL) lighter optimization required for each
elements type of sample.
0005270020.INDD 7 03-01-2022 17:50:15

Analytical Spot Size

5E22 100 at%
STEM/ STEM/
ICP Techniques
Auger SEM/ Raman XPS/ XRD XRR
EELS EDS Ellipsometry
1E22 EDS ESCA 10 at%
1E21 FTIR 1 at%

Atom
TGA/DTA/DSC
GPC
Probe
1E20 XRF 0.1 at%
DHEM
NMR
RBS
1E19 SEM-CL LIBS 100 ppm
Detection Range
Atoms/cm3
Ph
1E18 10 ppm
ys
GC-MS, LC-MS
ica
Ph
IGA
TXRF
IC
l li
ys
Ph
1 ppm
m
ica
1E17
ys
LA-
it
l li
fo
ica
ICPMS
r0
l li
it
.3
TOF-SIMS
m
100 ppb
or
1E16
nm
it
3n
fo
Elemental information
sa
r3
m
0n
sa
pl
1E15 Imaging information 10 ppb

m
in
pl
g
sa
Elemental and imaging information

in
de
m
pt
pl
de
Physical and/or optical properties

h
in
1E14 1 ppb
pt
Dynamic SIMS
g
ETV-ICP-OES
h
de
Thickness and density information

pt
h
1E13 Chemical bonding/molecular/structural information 100 ppt
GDMS
Electrical (active dopant and mobility) information Bulk
© 1995-2019 Eurofins EAG Materials Science. All Rights Reserved. Techniques
1E12 10 ppt
0.1 nm 1 nm 10 nm 100 nm 1 µm 10 µm 100 µm 1 mm 1 cm
TEM/STEM
EBSD
SEM
AFM
FIB
Imaging Techniques
Nanoindentation
EBIC
OP
RTX
Figure 1.1 A concise visual reference of most of the ring analytical techniques to compare the detection
limits and analytical resolutions for materials characterization. Source: Reproduced from Ref. [3] with the
kind permission and copyright of © Eurofins Scientific (www.eurofinseag.com).
In contrast to traditional AAS that can detect only one element at a time, ICP-MS instruments
have ability to measure all the elements present in the sample material even at once. However,
advanced AAS systems (AnalyticJena) are also available, which is of the scan variety (not inde-
pendent hollow cathode lamp) and can measure the elements sequentially (http://www.analytik-
jena.com). ICP-MS is widely used in forensic and biomedical science, in particular toxicology [3].
Depending on the specific parameters in the patient, the collection of samples taken for the analy-
sis process can vary from blood, serum, plasma, urine, to even packed red blood cells. This instru-
ment is also used in the environmental field. The applications include testing of water samples in
the soil for municipalities water and for industrial purposes.
The ICP-MS instrument should be free of obstruction. Even the smallest obstruction can disturb
the flow of the sample, which can clog the sample tips within the spray chamber. Also, high con-
centrations of NaCl in samples such as sea or ocean water can lead to obstruction. These blockages
can be overcome by dilution of the samples wherever a high concentration of salt has been observed
and compensated for. This process comes at the cost of detection limits. ICP-MS has been used for
glass analysis in forensic applications [3, 4]. It is capable of tracing the elements on the glass. The
elements detected on the glass can be utilized in order to match the sample materials observed at
the crime scene.
Laser ablation ICP-MS (LA-ICP-MS) uses a high-power pulsed laser beam (typically ns) to ablate
a small amount of material (picograms to femtograms) from the surface of the sample [3]. A plume
of atomic particles and ions are generated which are then carried to an ICP-MS detector with the
help of a constant flow of argon (Ar) or helium (He) gas. The sample is subsequently ionized in an
IC plasma, and its atomic species are transported in the form of ions, which are further separated
and analyzed using their mass/charge ratio. It is used to measure major and trace elemental com-
position of samples at the level of parts-per-billion (ppb). It is considered a versatile technique due
to its high analytical performance for various kinds of unprepared solid samples. A very small
amount of the sample (solid and liquid) quantities (picograms to femtograms) is sufficient to pro-
duce highly sensitive results up to the ppb level, depending on the measurement system. The laser
beam can be focused up to 5–200 μm range and thus allows a single spot analysis and line scanning
over the surface of the samples. It is recognized as a good analytical technique that can be used for
the analysis of a variety of sample materials detected in forensic applications [3]. It has already
proven its potential in the forensic analysis of bone, tooth, car paint, printing ink, metals, glasses,
trace fingerprints, soil, and paper fields [3].
A comparative study of LA-ICP-MS and micro-XRF by Gholap et al. [12] was performed in order
to compare their detection limits and spatial resolution. The experiment for elemental imaging was
performed on Daphnia magna, which is typically used as an indicator of aquatic ecosystem health
and is ascribed as a model organism in ecotoxicology. The authors used sections of the freshwater
crustacean D. magna (typical thickness of 10-20 μm) for the analysis and obtained the elemental
localization of elements in particular Ca, P, S, and Zn which allowed elemental correlation with
the tissues. The authors plotted the RGB maps (as shown in Figure 1.2) to conceptualize the simul-
taneous presence of metallic elements in the sample. Figure 1.2 shows the RGB representation of
the distribution of Ca, Fe, P using μ-XRF and Ca, P, Zn using LA-ICP-MS in the sagittal and dors-
oventral parts of D. magna. The results reveal the concomitant presence of Ca/P in thoracic
appendages, P/Zn in the gut and Ca/Zn in the exoskeleton. The co-existence of Ca/P and Zn/P is
ascribed to the formation of intracellular and membrane-bound phosphate granules, which can be
a reason for the storage of metallic ions Ca2+ and Zn2+ in living tissues [12, 13]. Both the tech-
niques provide comparable limits of detection (LOD) for Ca and P which validated the imaging
P P
Ca Fe Ca Zn
C
140 × 25 µm
D
B B
C
95 × 25 µm SAGITTAL DORSOVENTRAL
MICRO-XRF LA-ICPMS
Figure 1.2 RGB representation of Ca, Fe, P (micro-XRF) and Ca, P, Zn (LA-ICP-MS) distribution in sagittal
and dorsoventral sections of Daphnia magna. The sagittal sections originate from different depths of the
organism. A: thoracic appendages; B: eggs; C: carapax; D: gut epithelium. Source: Reproduced from Gholap
et al. [12] with permission from Elsevier.
results. LA-ICP-MS was found to be sensitive in determining Zn (LOD 20 ppm, 15 μm spot size) in
D. magna, but the detection power of μ-XRF was found inadequate. On the other hand, LA-ICP-MS
was found inadequate for the distribution analysis of S, which could be better examined and visu-
alized using μ-XRF (LOD 160 ppm, five seconds life time).
Finally, they were able to conclude that the use of a super-cell significantly reduced the volume
of ablation chamber, which significantly improved the lateral resolution. The spatial resolution of
LA-ICP-MS was found to be better than that of μ-XRF, however wash-out effects and spikes mar-
ginally disturbed the quality of image. μ-XRF provided the elemental distribution for S and LA-
ICP-MS gave the elemental distribution of Zn and thus both the techniques can be used in a
complementary manner. Synchrotron radiation in μ-XRF can be used to obtain better detection
power comparable to or higher than LA-ICP-MS. It can also be useful in order to obtain better
spatial resolution. Further, the application of LA-ICP-MS could be expanded to obtain 3D-elemental
distribution of elements as well as isotopes within biological tissues [14].
1.3.3 Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)

ICP-AES is an analytical technique that allows researchers to ascertain the quantitative bulk ele-
mental composition of samples in solid, liquid, powder, and suspension forms [3, 4]. In this
method, samples are digested using a mixture of acids in a closed microwave system and retains
potentially volatile analyte species. The prepared solution is then nebulized into the core of IC
argon plasma, and a temperature of nearly 9000 K is established. At this high temperature, the
nebulized solution is vaporized and the analyte species are atomized, ionized, and thermally
excited. After that, the analyte species are detected using an optical emission spectrometer. The
AES spectrometer measures the intensity of radiation emitted by the specific element present in
the sample which is proportional to the concentration of that element. Finally, the concentrations
of the elements are determined using the standard calibration curve method, which is mostly used
in cases where standards are unavailable.
The ICP-AES method is also used to determine the content of metals in wines and alcoholic
beverages, heavy metals such as arsenic (As) in food stuffs, and trace elements in complex biomol-
ecules such as proteins. It is capable to determine traces of oil additives which further indicate the
service life left for the motor oil. It is capable to detect Li (Z = 3) to U (Z = 92) except gases, halo-
gens, low contents of P and S. However in XRF, P and S can be easily determined and quantified.
The combined use of ICP-AES and ICP-MS is very powerful and gives highly accurate and precise
results for a broad range of elements from the major (percentage, %) to trace levels (typically
sub ppb) [3].
The disadvantage of this technique is that the emission spectra are complex and subject to spec-
tral interferences for some elements. Matrix effects also create many challenges to quantifying the
elements of interest. Some of the lither elements such as C, H, N, O, and halogens cannot be deter-
mined using this technique. Some elements cannot be detected by ICPs but often are subject to be
analyzed exclusively by XRF such as S, Br, and Cl.
In case of the limits of quantification equal to or above 1 ppm (μg/g), or where non-destructive
analysis is required, XRF is the most popular and attractive technique to analyze the solid samples,
powders, oils, and slurry samples. As opposed to ICP-AES, ICP-MS, and AAS, it does not require
sample dissolution or digestion and allows essentially non-destructive analysis. In that case, XRF
ensures accurate and reliable results by avoiding the potential for inaccuracies caused by incom-
plete dissolution and large dilutions.
1.3.4 Ion Chromatography (IC)

Ion Chromatograhy (IC) is a versatile analytical technique that is generally applied to detect posi-
tive and negative ions. It utilizes an ion’s intrinsic affinity for both an “eluent” (typically buffered
water) and a “stationary phase” (porous solid support with charge-bearing functional groups)
[3, 7]. It works on any kind of charged molecule, including large proteins, small nucleotides and
amino acids.
There are two types of IC, cation-exchange and anion-exchange. Cation-exchange chromatogra-
phy is used for positively charged molecules (pH < pI). The stationary phase is negatively charged
and positively charged molecules are loaded to get attracted toward it. In anion-exchange chroma-
tography, the stationary phase is positively charged and negatively charged molecules (pH > pI) are
loaded to get attracted toward it. It is used in water analysis, protein purification, and quality con-
trol. Some of the ideal uses of IC include the analysis of food products and beverages, aqueous
solutions such as water, and water-extractable surfaces. It is more useful for analysis for quality
assurance and control, purification of charged molecules, quantitative analysis of ions such as
cations (Li, Na, K, Mg, Ca, NH4+), anions (bromide, fluoride, nitrate, phosphate, sulfate, etc.),
secondary amines, chlorite, sulfate, iodide, bromated, etc. Some of the advantages and limitations
are tabulated in Table 1.2.
IC has industrial applications too. Its main advantages in this sector include good precision and
accuracy, reliability, high separation efficiency, high selectivity, good speed, and low operating
cost. Applications of IC particularly in the field of pharmaceutical industry are being developed.
These applications are typically focused on the determination of detection limits in the field of
pharmaceuticals. The detection limits corresponding to oxalates, sulfamates, sulfates, iodide,
phosphate, and electrolytes like sodium and potassium can be determined too. The IC can also be
used for the analysis of drugs having pharmaceutical importance for the development of products
with quality control testing. It can be used in pharmaceutical drugs in tablet or capsule form for the
determination of the actual dosage of the drugs that can be dissolved within some time. IC can also
be utilized for the detection as well as quantification of the inactive or undesirable ingredients that
are being used in the pharmaceutical formulations. Sugar and associated alcohol have been
detected in such formulations with the help of IC as they are easily resolved in an ion column due
to having polar groups. IC can also analyze the impurities present in the drug substances and prod-
ucts. Impurities in the drug can be easily estimated and help to provide an intuition for the mini-
mum and maximum dosage of drugs needed by a person on daily basis.
Table 1.2 Advantages and limitations of ion chromatography (IC) [3, 7].
Advantages Limitations
●● Small sample quantity required. ●● Buffer requirement

●● Rapid determination of anions and cations ●● Determination of only ionic analytes
(inorganic as well as organic) ●● Identification of peaks based on a retention time
●● Sensitivity: μg/l level match to a standard solution
●● Analysis of ionic species ●● Small change in pH greatly alters binding profile of
●● Stability of the separator columns stationary phase and ion states
●● Samples applied to the IC under conditions of low
ionic strength and controlled pH
●● Resistant to salt-induced corrosion
1.3.5 Laser-Induced Breakdown Spectroscopy (LIBS)

LIBS is a recently developed and rapid spectrochemical analysis technique and used to measure
the relative elemental concentrations and their distribution within the samples [3, 5, 15–18]. The
schematic diagram of LIBS is shown in Figure 1.3. LIBS uses a high-power pulsed laser beam that
atomizes as well as excites the sample material. The creation of plasma happens only when the
concentrated laser beam passes a threshold of power for an optical breakdown that is dependent of
the target material and environment. LIBS can analyze any matter, be it solid, liquid or gas. Since
all elements emit light of characteristic frequencies when excited to high temperatures, LIBS can
detect all elements. It is limited by the power of the laser, sensitivity of the instrument, wave-
lengths corresponding to the spectrograph, and the detector. LIBS can be employed for the deter-
mination of the relative amount of element constituents and even impurities present in the sample
material if the composition of the material is already known. Practically, it has been observed that
the detection limits depend upon the temperature corresponding to the plasma excitation, the
window used for the collection of light, and the line strength for the observed transitions.
LIBS is more advantageous for depth analysis due to its refocused capability on the same loca-
tion of a sample surface and its ability to provide depth profiling at a resolution of hundreds of
nanometers per pass. In addition, the laser beam can be focused from 20 to 200 μm and thus allows
the laser beam to scan across the whole sample surface that provides spatially resolved elemental
mapping. It can detect nearly all the naturally occurring element down to down to ppm level,
depending on the sample matrix.
Laser Synchronization Computer

Trigger Camera
Laser beam (266 nm/1064 nm)
Laser Source Beam
Power bending
Supply (Nd: YAG Laser)
mirror
Focusing lens
Long Optical
Laser Induced
fiber
Plasma
High Resolution
ICCD Spectrometer
ICCD
Controller
Collecting
Sample optics
Chamber
Gas
Controller Unit Sample
Gas Data Recording System Translational and

Gas Flow
Cylinder Rotational Stage
Figure 1.3 A schematic setup of laser-induced breakdown spectroscopy (LIBS).

LIBS can even detect halogen-based agents. The detection of heavy and toxic elements such as
lead (Pb) and mercury (Hg) in soil and plants can be determined by employing a field-portable
LIBS system. It has been observed that the analysis of the spectral emission of aluminum and alu-
minum oxides arises from the bulk aluminum in distinct bath gases can also be possible. It is used
for kinetic modeling of LIBS plumes. It is also used to detect and discriminate various materials
belonging to the category of explosives, geological, plastics, landmines, chemical as well as biologi-
cal warfare agents.
LIBS and XRF alike are generally used for positive material identification (PMI). For most of the
applications, LIBS provides the same information as XRF, just using a laser source instead of radia-
tion. But, in certain circumstances, one is better to use over the other. For example, handheld XRF
systems are easy to use compared with handheld LIBS systems [3, 5]. Handheld XRF gives more
precise results as compared with its LIBS counterpart. XRF is better than LIBS for the trace detec-
tion of elements below 0.1%. XRF can detect some heavier elements easily as compared to LIBS
such as tungsten (W) which LIBS cannot vaporize. Handheld XRF is useful for testing a wider
variety of elements however, LIBS is more suitable for the testing of lighter elements (C, H, N, O,
B, and Li). Handheld LIBS is faster than XRF at testing. Also, the cost of a handheld XRF device is
less than handheld LIBS.
Technically, the LIBS method is quite similar to other laser-based analytical methods, where
most of the hardware setup is same. LIBS can also be combined with Raman spectroscopy, and
fluorescence (Laser induced fluorescence : LIF) [3, 5, 19]. Now-a-days, the manufactured devices
combine these techniques in a single instrument, thereby allowing the atomic, molecular and
structural characterization of a specimen, thus giving a deeper insight into the physical proper-
ties of it.
1.3.6 Proton-Induced X-Ray Emission (PIXE)

PIXE technique involves the detection of X-rays emitted from the sample due to the bombard-
ment of the sample with high energy ions [3, 20]. Different types of excitation beams produce
X-rays with energies which are characteristic of the elements present in the sample. Electron
excitation in an electron microprobe (SEM) gives rise to EDXRF or WDXRF, which depends on
the X-ray dispersion and detection mode. Charged particle beams of He2+ or H+ gives rise to
PIXE spectroscopy. In all these spectroscopic methods, the excitation beam removes a core elec-
tron, which creates a vacancy, causing outer shell electrons to jump down to fill the inner shell
vacancy. Thus, X-rays are emitted with specific energies which are characteristic to the elements
present in the sample. This accessory is very beneficial for identifying heavy elements by
Rutherford backscattering spectrometry (RBS) [3]. These heavy elements show only small differ-
ences in RBS backscattered energies because of their similar masses, but there are distinct differ-
ences in their PIXE spectral studies. Being a non-destructive analytic technique, PIXE offers
signal levels similar to its electron beam counterparts, but provides better signal/background
ratios. In electron spectroscopy, the background arises from bremsstrahlung and this is abso-
lutely absent in PIXE due to the use of He2+ or H+ ions [3]. PIXE can analyze insulating samples
which cannot be detected by electron-induced spectroscopy. However, some limitations are also
present, such as the analysis area is limited to 1–2 mm and useful information can be obtained to
only ~1 μm depth of samples with good accuracy.
Recent developments in PIXE use tightly-focused beams to enhance the ability of microscopic
analysis. This technique is called micro-PIXE [3, 21] and is used for determining the distribution
of trace elements in a wide range of samples. A similar technique, particle-induced gamma-ray
Table 1.3 Advantages and limitations of SEM-EDS method [3, 8].
Advantages Limitations
Quick identification of elements Size restriction which requires cutting of

Provides high-resolution imaging the samples
Excellent depth of field for 3D appearance of the Need to etch planar samples for contrast
specimen image (~100X that of Optical microscopy) Ultimate resolution is a strong function of
Versatile platform and supports many other the sample chemistry and stability in the
analytical techniques electron beam
Possibility of imaging of insulating and hydrates
samples in low vacuum mode
emission (PIGE) [3, 22] is used to detect some light elements. PIXE is a better method than the
traditional XRF techniques as it can detect the elements and their ratio too. On the other hand,
SRXRF is better than PIXE.
1.3.7 Scanning Electron Microscopy–Energy Dispersive X–Ray Spectroscopy (SEM-EDS)

SEM provides high-resolution images of the sample surfaces [1–3, 8, 9]. It is the most popular ana-
lytical tool as it can quickly provide the detailed and good quality images of the sample. It can be
coupled to an auxiliary EDS detector and provide elemental identification for most of the elements
of the entire periodic table. SEM-EDS is used where optical microscopy cannot provide sufficient
image resolution of sufficient magnification. It also produces detailed surface topography images
[3, 8]. Some of the advantages and limitations of SEM-EDS are tabulated in Table 1.3.
In SEM-EDS, the sample is scanned by the electron-beam of the microscope and the different
interactions are employed to obtain the images using secondary or backscattered electrons.
Additionally, elemental analysis of the samples is performed by using the excitation of fluores-
cence radiation. Therefore, using the information obtained by images and elemental contents, the
samples can be compared directly.
From Table 1.1, it is clear that μ-XRF and SEM-EDS possess different spatial resolutions and also
have some differences for elemental detection [3, 8]. However, the main differences exist in terms
of specific sample handling and some analytical capabilities.
1.3.7.1 Differences in XRF and SEM-EDS (Sample Handling, Experimental Conditions,

Sample Stress, and Excitation Sources)
There are some differences in XRF and SEM-EDS such as sample handling, experimental condi-
tions, sample stress, and excitation sources [1, 8]. Sample handling processes for XRF are quite
easy when compared to those used for SEM-EDS. Unlike for SEM-EDS, the electrical conductiv-
ity requirement of the sample is not needed for μ-XRF [3, 8]. In SEM analysis, the excitation is
carried out by electrons which transport electrical charges to the sample. These charges have to
be removed, and therefore the sample must be electrically conductive. Otherwise, image quality
and quantification are affected. In cases of a non-electrical conducting samples, coating by
carbon (C) or gold (Au) of the samples is quite important. In SEM analysis, this is the only addi-
tional effort needed for analyzing samples such as glass, minerals, and plastics. On the other
hand, these kinds of samples are analyzed directly using μ-XRF, which reduces the sample prep-
aration time significantly.
During SEM experimentation, sample surface must be of a very high quality due to the low pene-
tration depth of electrons inside the materials. Thus, polishing of the sample needs more attention
for better results. On the contrary, this kind of sample treatment is not required for μ-XRF analysis.
μ-XRF analysis can also be performed in air or pre-vacuum. Due to this advantage of μ-XRF,
vacuum-sensitive materials (organic samples) are easily analyzed [3, 8]. Also, simple liquids or wet
samples such as pastes, slurry, etc. can also be analyzed quickly. This makes μ-XRF applicable for
a wide range of materials. Sometimes a vacuum condition is needed for μ-XRF just to avoid the
absorption of the fluorescence radiation in air, and thus 10–50 mbar pressures are sufficient. In
SEM analysis, the absorption of electrons must be avoided and thus pressures required are in the
range down to 0.01 mbar.
Some differences also exist between the excitation by X-rays in XRF and by electrons in SEM
with respect to the sample stress. The absorption of electrons by the target material is accompanied
by a higher impact of energy into the material that heats up the material and stresses it. Sometimes,
it damages the materials. Contrarily, the absorption of X-rays did not produce high energy impact
into the sample, and thus the heating effect is negligible, which reduces the sample stress.
Therefore, higher excitation intensities can be used for μ-XRF analysis.
A comparison of the analytical performances of XRF and SEM-EDS indicates the differences for
sensitivity particularly for analysis of traces. To analyzed trace elements, the sensitivity mainly
depends on the peak/background ratio [23]. For electron excitation, the background intensity is
higher because of the bremsstrahlung of the electron beam. The spectral background for X-ray
excitation is mainly due to the scattering of the bremsstrahlung of the tube on the sample. The
other factor that influences peak/background ratio is the peak-intensity which is determined by
the quantity of the element and also by its excitation efficiency i.e. the excitation conditions and
the cross-sections for the excitation. Figure 1.4 clearly shows the excitation efficiency for electrons
and X-rays that indicates that the cross section of electrons and X-rays for exciting the K-shell
depends on the atomic numbers of the atoms [23]. This diagram reveals the high efficiency (high
cross section) of electron excitation for lighter elements. In electron microscopes, lighter elements
can easily be detected; even boron (B) or beryllium (Be) can be measured. However, heavy ele-
ments can be detected easily by excitation with X-rays with better efficiency. Therefore, all the
elements with atomic number greater than 20 (such as Ca) exhibit higher peak intensities and
better sensitivities with X-ray excitation [23]. This also results in better LODs in the case of heavy
elements, as demonstrated in Figure 1.5 [23].
Figure 1.4 K-shell cross sections for 10,000

electron and X-ray irradiation. Here X 5 keV 20 keV
K-X-ray emission cross section
X-denotes the emission due to X-rays.

Source: Reproduced from Haschke and 1000
Boehm [23] with permission from Elsevier. 60 keV
(barn)
100
10
10 keV
50 keV
1
0 20 40 60
Atomic number
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cause o’t to ony man, though I whiles think it wad be naething to me,
that’s sae weel used till’t mysel.’
“‘Helen,’ said I, ‘when did Willie Meldrum find opportunities to
gain your heart? I never saw him in the house in my life.’
“‘Oh, sir!’ said she, ‘gin I could hae bidden in the house, he wad
never hae seen me either; but I was forced to walk out wi’ the bairns,
and there was nae place sae quiet and out o’ the gate, but Willie was
sure to find me out. If I gaed down the burn, Willie was aye fishing; if
I gaed up the loan, there was aye something to be dune about the
kye. At the kirk door, Willie was aye at hand to spier for your honour,
and gie the bairns posies; and after our sair distress, when I was little
out for mony a day, I couldna slip out ae moonlight night, to sit a
moment upon Jeanie’s grave, but Willie was there like a ghaist aside
me, and made my very heart loup to my mouth!’
“‘And do you return his good-will, Helen?’ said I, gravely.
“‘Oh, sir,’ said the poor thing, trembling, ‘I darena tell you a lie. I
tried to be as proud and as shy as a lassie should be to ane abune her
degree, and that might do sae muckle better, puir fallow! I tried to
look anither gate when I saw him, and mak mysel deaf when he
spoke o’ his love; but oh! his words were sae true and kindly, that I
doubt mine werena aye sae short and saucy as they sud hae been. It’s
hard for a tocherless, fatherless lassie to be cauldrife to the lad that
wad tak her to his heart and hame; but oh! it wad be harder still, if
she was to requite him wi’ a father’s curse! It’s ill eneuch to hae nae
parents o’ my ain, without makin’ mischief wi’ ither folk’s. The auld
man gets dourer and dourer ilka day, and the young ane dafter and
dafter—sae ye maun just send me aff the country to some decent
service, till Willie’s a free man, or a bridegroom.’
“‘My dear Helen,’ said I, ‘you are a good upright girl, and I will
forward your honest intentions. If it be God’s will that Willie and you
come together, the hearts of men are in His hand. If otherwise, yours
will never at least reproach you with bringing ruin on your lover’s
head.’
“So I sent Helen, Mr Francis, to my brother’s in the south country,
where she proved as great a blessing and as chief a favourite as she
had been with us. I saw her some months afterwards; and though her
bloom had not returned, she was tranquil and contented, as one who
has cast her lot into the lap of Heaven.
“Well, to make a long story short, Willie, though he was
unreasonable enough, good, worthy lad as he is, to take in dudgeon
Helen’s going away (though he might have guessed it was all for his
good), was too proud, or too constant, to say he would give her up, or
bind himself never to marry her, as his father insisted. So the old
man, one day, after a violent altercation, made his will, and left all
his hard-won siller to a rich brother in Liverpool, who neither
wanted nor deserved it. Willie, upon this quarrel, had left home very
unhappy, and stayed away some time, and during his absence old
Blinkbonnie was taken extremely ill. When he thought himself dying,
he sent for me (I had twice called in vain before), and you may be
sure I did my best not to let him depart in so unchristian a frame
towards his only child. I did not deny his right to advise his son in
the choice of a wife; but I told him he might search the world before
he found one more desirable than Helen, whose beauty and sense
would secure his son’s steadiness, and her frugality and sobriety
double his substance. I told him how she had turned a deaf ear to all
his son’s proposals of a clandestine marriage and made herself the
sacrifice to his own unjust and groundless prejudices. Dying men are
generally open to conviction; and I got a fresh will made in favour of
his son, with a full consent to his marriage honourably inserted
among its provisions. This he deposited with me, feeling no great
confidence in the lawyer who had made his previous settlement, and
desired me to produce it when he was gone.
“It so happened that I was called to a distance before his decease,
and did not return till some days after the funeral. Willie had flown
home on hearing of his father’s danger, and had the comfort to find
him completely softened, and to receive from his nearly speechless
parent many a silent demonstration of returned affection. It was,
therefore, a doubly severe shock to him, on opening the first will (the
only one forthcoming in my absence), to find himself cut off from
everything, except the joint lease of the farm, and instead of five
thousand pounds, not worth a shilling in the world. His first
exclamation, I was told, was, ‘It’s hard to get baith scorn and skaith—
to lose baith poor Helen and the gear. If I had lost it for her, they
might hae ta’en it that likit!’
“About a week after, I came home and found on my table a letter
from Helen. She had heard of Willie’s misfortune, and in a way the
most modest and engaging, expressed herself ready, if I thought it
would still be acceptable, to share his poverty and toil with him
through life. ‘I am weel used to work,’ said she, ‘and, but for you, wad
hae been weel used to want. If Willie will let me bear a share o’ his
burden, I trust in God we may warsle through thegither; and, to tell
you the truth,’ added she, with her usual honesty, ‘I wad rather
things were ordered as they are, than that Willie’s wealth should
shame my poverty.’
“I put this letter in one pocket, and his father’s will in the other,
and walked over to Blinkbonnie. Willie was working with the manly
resolution of one who has no other resource. I told him I was glad to
see him so little cast down.
“‘Sir,’ said he, ‘I’ll no say but I am vexed that my father gaed to his
grave wi’ a grudge against me, the mair sae, as when he squeezed my
hand on his death-bed I thought a’ was forgotten. But siller is but
warld’s gear, and I could thole the want o’t, an’ it had nae been for
Helen Ormiston, that I hoped to hae gotten to share it wi’ me. She
may sune do better now, wi’ that bonnie face and kind heart o’ hers!’
“‘It is indeed a kind heart, Willie,’ answered I: ‘if ever I doubted it,
this would have put me to shame!’——So saying, I reached him the
letter, and oh, that Helen could have seen the flush of grateful
surprise that crossed his manly brow as he read it! It passed away,
though, quickly, and he said, with a sigh, ‘Very kind, Mr Monteith,
and very like hersel; but I canna take advantage o’ an auld gude will,
now that I canna reward it as it deserves!’”
‘And what if ye could, Willie?’ said I, ‘as far, at least, as worldly
wealth can requite true affection? There is your father’s will, made
when it pleased God to touch his heart, and you are as rich a man as
you were when Helen Ormiston first refused to make you a beggar.’
“Willie was not insensible to this happy change in his prospects;
but his kind heart was chiefly soothed by his father’s altered feelings,
and at the honourable mention of Helen’s name he fairly began to
greet.
“The sequel is easily told; but I think the jaunt I made to
Tweeddale with Willie, to bring back Helen Ormiston in triumph,
was the proudest journey of my life.
“A year ago I married them at the manse, amid much joy, but
abundance of tears in the nursery. To-day, when, according to an old
promise, I am to christen my name-son Charlie, I expect to be fairly
deaved with the clamorous rejoicings of my young fry, who, I verily
believe, have not slept this week for thinking of it. But” (pulling out
his watch), “it is near four o’clock: sad quality hour for Blinkbonnie!
The hotch-potch will be turned into porridge, and the how-towdies
burnt to sticks, if we don’t make haste!”
I wish, my dear reader, you could see the farm of Blinkbonnie,

lying as it does on a gently sloping bank, sheltered from the north by
a wooded crag or knoll, flanked upon the east by a group of venerable
ashes, enlivened and perfumed on the west by a gay luxuriant
garden, and open on the south to such a sea-view, as none but
dwellers on the Firth of Forth have any idea of. Last Saturday, it was
the very beau ideal of rural comfort and serenity. The old trees were
reposing, after a course of somewhat boisterous weather, in all the
dignity and silence of years. The crows, their usual inhabitants,
having gone on their Highland excursion, those fantastic interlopers,
Helen’s peacocks (a present from the children at the manse), were
already preparing for their “siesta” on the topmost boughs. Beneath
the spreading branches the cows were dreaming delightfully, in
sweet oblivion of the heats of noon. In an adjoining paddock,
graceful foals, and awkward calves, indulged in their rival gambols;
while shrieks of joy from behind the garden hedge, told these were
not the only happy young things in creation.
We deposited our horses in a stable, to whose comforts they bore
testimony by an approving neigh, and made our way by a narrow
path, bordered with sweet-brier and woodbine, to the front of the
house. Its tall, good-looking young master came hastily to meet us,
and I would not have given his blushing welcome, and the bashful
scrape that accompanied it, for all the most elaborate courtesies of
Chesterfield.
No sooner were our footsteps heard approaching, than out poured
the minister’s whole family from the little honey-suckled porch, with
glowing faces and tangled hair, and frocks, probably white some
hours before, but which now claimed affinity with every bush in the
garden.
Mrs Monteith gently joined in the chorus of reproaches to papa for
being so late; but the look with which she was answered seemed to
satisfy her, as it usually did, that he could not be in fault. We were
then ushered into the parlour, whose substantial comforts, and
exquisite consistency, spoke volumes in favour of its mistress.
Opulence might be traced in the excellent quality of the homely
furniture—in the liberal display of antique china (particularly the
choice and curious christening-bowl)—but there was nothing
incongruous, nothing out of keeping, nothing to make you for a
moment mistake this first-rate farmhouse parlour for a clumsy, ill-
fancied drawing-room. A few pots of roses, a few shelves of books,
bore testimony to Helen’s taste and education; but there were
neither exotics nor romances in the collection; and the piece of
furniture evidently dearest in her eyes was the cradle, in which
reposed, amid all the din of this joyous occasion, the yet
unchristened hero of the day. It is time to speak of Helen herself, and
she was just what, from her story, I knew she must be. The actors, in
some striking drama of human life, often disappoint us by their utter
dissimilitude to the pictures of our mind’s eye, but Helen was
precisely the perfection of a gentle, modest, self-possessed Scottish
lassie,—the mind, in short, of Jeanie Deans, with the personal
advantages of poor Effie. Her dress was as suitable as anything else.
Her gown, white as snow, and her cap of the nicest materials, were
neither of them on the pattern of my lady’s; but they had a matronly
grace of their own, worth a thousand second-hand fashions; and
when Helen, having awakened her first-born, delivered him, with
sweet maternal solicitude, into the outstretched arms of the
minister’s proud and favoured youngest girl, I thought I never saw a
picture worthier the pencil of Correggio. It was completed, when,
bending in all the graceful awkwardness of a novice over the group,
Willie received his boy into his arms, and vowed before his pastor
and his God to discharge a parent’s duty, while a parent’s transport
sparkled in his eyes.
I have sat, as Shakspeare says, “at good men’s feasts ere now”—
have ate turtle at the lord mayor’s and venison at peers’ tables, and
soufflés at diplomatic dinners—have ate sturgeon at St Petersburg,
and mullet at Naples; mutton in Wales, and grouse in the Highlands;
roast-beef with John Bull, and volauxvents at Beauvilliers’; but I
have no hesitation in saying that the hotch-potch and how-towdies of
Blinkbonnie excelled them all. How far the happy human faces of all
ages round the table contributed to enhance the gusto, I do not
pretend to decide; but I can tell Mr Véry that, among all his
consommés, there is nothing like a judicious mixture of youth and
beauty, with manliness, integrity, and virtue.—Blackwood’s
Magazine.
A SCOTTISH GENTLEWOMAN OF THE LAST
CENTURY.
By Susan Edmonstone Ferrier.
“Though last, not least of nature’s works, I must now introduce

you to a friend of mine,” said Mr Douglas, as they bent their steps
towards the Castlehill of Edinburgh. “Mrs Violet Macshake is an aunt
of my mother’s, whom you must often have heard of, and the last
remaining branch of the noble race of Girnachgowl.”
“I am afraid she is rather a formidable person, then?” said Mary.
Her uncle hesitated.
“No, not formidable,—only rather particular, as all old people are;
but she is very good-hearted.”
“I understand; in other words, she is very disagreeable. All ill-
tempered people, I observe, have the character of being good-
hearted, or else all good-hearted people are ill-tempered—I can’t tell
which.”
“It is more than reputation with her,” said Mr Douglas, somewhat
angrily; “for she is, in reality, a very good-hearted woman, as I
experienced when a boy at college. Many a crown-piece and half-
guinea I used to get from her. Many a scold, to be sure, went along
with them; but that, I daresay, I deserved. Besides, she is very rich,
and I am her reputed heir; therefore gratitude and self-interest
combine to render her extremely amiable in my estimation.”
They had now reached the airy dwelling where Mrs Macshake
resided, and having rung, the door was at length most deliberately
opened by an ancient, sour-visaged, long-waisted female, who
ushered them into an apartment, the coup d’œil of which struck a
chill to Mary’s heart. It was a good-sized room, with a bare
sufficiency of small-legged dining-tables, and lank hair-cloth chairs,
ranged in high order round the walls. Although the season was
advanced, and the air piercing cold, the grate stood smiling in all the
charms of polished steel; and the mistress of the mansion was seated
by the side of it in an arm-chair, still in its summer position. She
appeared to have no other occupation than what her own
meditations afforded; for a single glance sufficed to show that not a
vestage of book or work was harboured there. She was a tall, large-
boned woman, whom even Time’s iron hand had scarcely bent, as
she merely stooped at the shoulders. She had a drooping snuffy nose,
a long turned-up chin, small, quick, gray eyes, and her face projected
far beyond her figure, with an expression of shrewd, restless
curiosity. She wore a mode (not a-la-mode) bonnet, and cardinal of
the same; a pair of clogs over her shoes, and black silk mittens on her
arms.
As soon as she recognized Mr Douglas, she welcomed him with
much cordiality, shook him long and heartily by the hand,—patted
him on the back,—looked into his face with much seeming
satisfaction; and, in short, gave all the demonstrations of gladness
usual with gentlewomen of a certain age. Her pleasure, however,
appeared to be rather an impromptu than a habitual feeling; for as
the surprise wore off, her visage resumed its harsh and sarcastic
expression, and she seemed eager to efface any agreeable impression
her reception might have excited.
“An’ wha thought o’ seein’ you e’noo?” said she, in a quick gabbling
voice; “what’s brought you to the toun? Are ye come to spend your
honest faither’s siller, ere he’s weel cauld in his grave, puir man?”
Mr Douglas explained, that it was upon account of his niece’s
health.
“Health!” repeated she, with a sardonic smile, “it wad mak a
howlet laugh to hear the wark that’s made about young fowk’s health
noo-a-days. I wonder what ye’re a’ made o’,” grasping Mary’s arm in
her great bony hand; “a wheen puir feckless windle-straes—ye maun
awa to England for yer healths. Set ye up! I wonder what came o’ the
lasses i’ my time, that but to bide hame? And whilk o’ ye, I sud like to
ken, will e’er live to see ninety-sax, like me?—Health! he! he!”
“You have not asked after any of your Glenfern friends,” said Mr
Douglas, hoping to touch a more sympathetic chord.
“Time eneugh—will ye let me draw my breath, man?—fowk canna
say a’ thing at ance. An’ ye but to hae an English wife, too?—A Scotch
lass wadna ser’ ye. An’ yer wean, I’se warran’, it’s ane o’ the warld’s
wonders—it’s been unco lang o’ comin’—he! he!”
“He has begun life under very melancholy auspices, poor fellow!”
said Mr Douglas, in allusion to his father’s death.
“An’ wha’s faut was that?—I ne’er heard tell the like o’t, to hae the
bairn kirsened an’ its grandfather deein’! But fowk are neither born,
nor kirsened, nor do they wad or dee as they used to do—a’thing’s
changed.”
“You must indeed have witnessed many changes,” observed Mr
Douglas, rather at a loss how to utter anything of a conciliatory
nature.
“Changes!—weel a wat, I sometimes wonder if it’s the same warld,
an’ if it’s my ain head that’s upon my shouthers.”
“But with these changes you must also have seen many
improvements?” said Mary, in a tone of diffidence.
“Impruvements!” turning sharply round upon her, “what ken ye
about impruvements, bairn? A bonnie impruvement to see tylors and
sclaters leevin’ whaur I mind Jukes and Yerls. An’ that great
glowerin’ New Town there,” pointing out of her windows, “whaur I
used to sit and look at bonnie green parks, and see the kye milket,
and the bits o’ bairnies rowin’ an’ tumblin’, an’ the lasses trampin’ in
their tubs;—what see I noo, but stane and lime, and stour and dirt,
and idle chiels, and dunket-out madams prancing.—Impruvements,
indeed!”
Here a long pinch of snuff caused a pause in the old lady’s
harangue; but after having duly wiped her nose with her coloured
handkerchief, and shook off all the particles that might be presumed
to have lodged upon her cardinal, she resumed:
“An’ nae word o’ ony o’ your sisters gaun to get men yet? They tell
me they’re but coorse lasses; an’ wha’ll tak ill-faured, tocherless
queans, when there’s walth o’ bonny faces an’ lang purses i’ the
market?—he, he!” Then resuming her scrutiny of Mary,—“An’ I’se
warran’ ye’ll be lookin’ for an English sweetheart too;—that’ll be
what’s takin’ ye awa to England!”
“On the contrary,” said Mr Douglas, seeing Mary was too much
frightened to answer for herself—“on the contrary, Mary declares she
will never marry any but a true Highlander—one who wears the dirk
and plaid, and has the ‘second sight.’ And the nuptials are to be
celebrated with all the pomp of feudal times; with bagpipes and
bonfires, and gatherings of clans, and roasted sheep, and barrels of
whisky, and”——
“Weel a wat an’ she’s i’ the right there,” interrupted Mrs
Mackshake, with more complacency than she had yet shown. “They
may ca’ them what they like, but there’s nae waddin’s noo. Wha’s the
better o’ them but innkeepers and chaise-drivers? I wadna count
mysel married i’ the hidlin’s way they gang aboot it noo.”
Mr Douglas, who was now rather tired of the old lady’s
reminiscences, availed himself of the opportunity of a fresh pinch to
rise and take leave.
“Ou, what’s takin’ ye awa, Archie, in sic a hurry? Sit doon there,”
laying her hand upon his arm, “an’ rest ye, and tak a glass o’ wine; or
maybe,” turning to Mary, “ye wad rather hae a drap broth to warm
ye. What gars ye look sae blae, my bairn? I’m sure it’s no cauld; but
ye’re just like the lave; ye gang a’ skiltin’ about the streets half-naked,
an’ then ye maun sit and birsle yersels afore the fire at hame.”
The wine being drunk, and the cookies discussed, Mr Douglas
made another attempt to withdraw, but in vain.
“Canna ye sit still a wee, man, an’ let me speir after my auld freens
at Glenfern? Hoo’s Grizzy, an’ Jacky, an’ Nicky?—aye working awa at
the pills and the drogs?—he, he! I ne’er swallowed a pill, nor gaed a
doit for drogs, a’ my days, an’ see an ony of them’ll run a race wi’ me
when they’re naur five score.”
Mr Douglas here paid her some compliments upon her
appearance, which were pretty well received; and added that he was
the bearer of a letter from his aunt Grizzy, which he would send
along with a roebuck and a brace of moor game.
“Gin your roebuck’s nae better than your last, atweel it’s no worth
the sending,—puir fushionless dirt, no worth the chewing; weel a
wat, I begrudged my teeth on’t. Your muirfowl was no that ill, but
they’re no worth the carrying; they’re dang cheap i’ the market e’noo,
so it’s nae great compliment. Gin ye had brought me a leg o’ good
mutton, or a caller sawmont, there would hae been some sense in’t;
but ye’re ane o’ the fowk that’ll ne’er harry yoursel wi’ your presents;
it’s but the pickle poother they cost you, an’ I’se warrant ye’re
thinking mair o’ your ain diversion than o’ my stamack when ye’re at
the shooting o’ them, puir beasts.”
Mr Douglas had borne the various indignities levelled against
himself and his family with a philosophy that had no parallel in his
life before; but to this attack upon his game he was not proof. His
colour rose, his eyes flashed fire, and something resembling an oath
burst from his lips, as he strode indignantly towards the door.
His friend, however, was too nimble for him. She stepped before
him, and breaking into a discordant laugh, as she patted him on the
back,—
“So, I see ye’re just the auld man, Archie,—aye ready to tak the
strumps, an ye dinna get a’thing yer ain way. Mony a time I had to
fleech ye oot o’ the dorts when ye was a callant. Div ye mind hoo ye
was affronted because I set ye doon to a cauld pigeon pie an’ a tanker
o’ tippenny, ae night to yer four-hours, afore some leddies? he, he,
he! Weel a wat, your wife maun hae her ain adoos to manage ye, for
ye’re a cumstarie chield, Archie.”
Mr Douglas still looked as if he was irresolute whether to laugh or
be angry.
“Come, come, sit ye doon there till I speak to this bairn,” said she,
as she pulled Mary into an adjoining bed-chamber, which wore the
same aspect of chilly neatness as the one they had quitted. Then
pulling a large bunch of keys from her pocket, she opened a drawer,
out of which she took a pair of diamond ear-rings.
“Hae, bairn,” said she, as she stuffed them into Mary’s hand; “they
belanged to your faither’s grandmother. She was a good woman, an’
had four and twenty sons and dochters, an’ I wuss ye nae waur fortin
than just to hae as mony. But mind ye,” shaking her bony finger,
“they maun a’ be Scots. Gin I thocht ye wad marry ony pock-puddin’,
fient hait wad ye gotten frae me. Noo, haud yer tongue, and dinna
deave me wi’ thanks,” almost pushing her into the parlour again;
“and sin’ ye’re gaun awa the morn, I’ll see nae mair o’ ye e’noo—so
fare ye weel. But, Archie, ye maun come an’ tak your breakfast wi’
me. I hae muckle to say to you;—but ye maunna be sae hard upon my
baps as ye used to be,” with a facetious grin to her mollified
favourite, as they shook hands and parted.—“Marriage: a Novel.”
THE FAITHLESS NURSE:
A LEGENDARY TALE OF THE GREAT
REBELLION.
Most of our readers who are citizens of “our own romantic town,”
are familiarly acquainted with the valley which, winding among the
Pentland Hills, forms the path by which the waters of Glencorse seek
their way to those of the more celebrated Esk. It has long been the
haunt of those “pilgrims of his genius” who loved to see with their
own eyes the sacred scene chosen by the Pastoral Poet of Scotland for
the display of lowly loves and rustic beauty; and it has now—alas the
day!—acquired attractions for spirits of a far different sort; and who
can see without a sigh the triumphs of art domineering over and
insulting the sweetest charms of nature? It is not, however, to visit
the stupendous and unseemly barrier which now chains up the gentle
waters of the burn, nor even to seek the summer-breathing spot
where Patie sung and Roger sighed, that we now request the
attendance of our readers; but simply to point out to their attention a
party of three individuals, who, on a still September evening, in the
memorable year 1644, might have been seen slowly riding up the
glen.
Two of the party were entitled in courtesy to be termed fair; but of
these twain, one would have been acknowledged lovely by the most
uncourteous boor that ever breathed. She had hardly reached the
earliest years of womanhood, ’tis true, and the peachy bloom that
mantled o’er her cheek showed as yet only the dawn of future
loveliness; but her fair brow, on which, contrary to the fashion—we
had almost said taste—of the times, her auburn locks danced
gracefully; the laughing lustre of her dark-blue eye, and the stinging
sweetness of her pouting lip, aided by an expression of indomitable
gentleness of heart and kindliness of manner, lent a witchery to her
countenance which few could gaze upon unmoved.
The other female had thrice the years of Lady Lilias Hay; but they
had not brought her one tithe of that maiden’s beauty, and what little
God had given her, she had, long ere the day we saw her first,
destroyed, by screwing her features into an unvarying cast of prim
solemnity, which, had she practised it, would have blighted the cheek
of Venus herself.
The “squire of dames” who accompanied the pair we have
described was also young, his chin as yet being guiltless of a hair. But
there was a firmness in his look, a dark something in his eye, that
bespoke his courage superior to his years; and a scar that trenched
his open brow showed that he had arrived at the daring, if not the
wisdom of manhood.
On the present occasion, however, it was not a feeling of
recklessness which characterised the demeanour of the youth. He
was thoughtful and abstracted, riding silently by the side of the
maiden, who more than once attempted to dispel the gloom which
hung over the gallant. It gave way, indeed, to the influence of her
gentle voice; but it was for a moment only, and the downcast eye and
contracted brow ever and anon returned when the accents of her
voice had ceased.
“Nay, prithee, cousin Maurice, do doff the visor of thy melancholy,
and let us behold thy merry heart unmasked. I could stake my little
jennet here to Elspeth’s favourite “baudrons,” that if Montrose
should meet thee in this moody temperament, he will rather promote
thee to a halter as a spy from the Committee of Estates, than to
honourable command befitting one who has bled beneath the eye,
and been knighted by the honour-giving hand of his royal master! Do
laugh with me a little.”
“Why, my dearest Lilias, you seem in higher spirits to-day than is
usual with you. Cannot the surety of our parting to-morrow, and the
uncertainty of our ever meeting again, throw even a passing cloud
over your gaiety?”
“Modestly put, my valiant cousin. I am well reminded of my
unbecoming conduct. It must, of course, be night with me when you,
bright sun of my happiness, shall have withdrawn your beams from
me.”
“Nay, banter me not, sweet Lily. Have you never known an hour
when the sweetest sights were irksome to the eye, and the softest
strains of music fell harshly on the ear?”
“Pshaw! if you will neither smile nor talk, of what use are you by a
lady’s side? What say you to a race? Yonder stands the kirk of Saint
Catherine. Will you try your roan that length? An you ride not so fast
now as you did from Cromwell at Longmarston Moor, I shall beat
you. Via!”
And so saying, the light-hearted girl gave rein to her snowy palfrey,
and flew up the glen toward the edifice she had mentioned, at a
speed which Maurice Ogilvy had some difficulty in equalling, and
which prevented him from overtaking her until she had reached the
gate.
All who have visited—and who has not?—Roslin’s “proud
chapelle,” are familiar with the legend of Sir William St Clair, and his
venturous boast to the Bruce, that he would find, on peril of his head,
a dog that would bring down the deer ere it could cross Glencorse
burn;—how the trusty hound did redeem his own credit and his
master’s life, by seizing the quarry in the very middle of the stream;—
and how, in gratitude to the gentle saint by whose intercession this
mighty feat was accomplished, he built a church on the bank of the
stream, and dedicated it to Saint Catherine of the Howe. This virgin
martyr was unfortunately no more successful than her sister saints in
protecting her mansions from the desolating zeal of the earlier
reformers. The church was destroyed by a fanatical mob, and nothing
now remains to record the kindness of Catherine, and the gratitude
of the “high Saint Clair,” but a few uneven grassy heaps of deeper
green than the surrounding verdure, and the name of the
neighbouring farm town, which is yet called Kirkton. At the time we
are at present writing of, however, the roofless walls of the building,
though gray with the ruin of a hundred years, were still almost
entire, and the cemetery then and long after continued to be used by
the neighbouring peasantry.
When Maurice reached the church, he found that the Lady Lilias
had dismounted. He too alighted, and sought her in the interior. She
was seated on a fallen stone, and the deep melancholy which now
shadowed her fair countenance was more in unison with the sombre
aspect of the place and of the hour, than he had expected to find it.
She arose at his approach, and addressed him.
“You have something to tell me, Maurice, and you wished to do it
alone. We have now an opportunity. What has befallen us?”
“Nay, fair Lily, why should you think so? Is not the thought that to-
morrow we must part of itself sufficient to dull my spirit and sadden
my countenance?”
“Pshaw! trifle not with me now. Your face has no secrets for one
who has conned its ill-favoured features so frequently as I have done.
Out with your secret! Elspeth will be with us forthwith.”
Maurice seemed for some moments undecided how he should act,
but at length, with a look of no little embarrassment, replied,—
“Sweet Lilias, you shall be obeyed. You can only laugh at me; and
thanks to your merry heart, that is a daily pastime of yours.”
“Nay, nay—say on; I will be as grave as Argyle.”
“Know then, that while I waited for you and Elspeth at the bottom
of the glen, a remarkable thing befell me. I had alighted, and while
Rupert was trying to pick a scanty meal among the bent, I flung
myself on the ground, and endeavoured to beguile the time by
thinking sometimes of you, and sometimes of King Charles.”
“How! sir cousin, I am not always the companion of your reveries,
it seems, then? Heigho! to think what a change a single day’s
matrimony has accomplished!”
“Ungenerous Lilias,” said Maurice, taking her hand, “listen to me.
Lifting my head accidentally, I was surprised to perceive a man and
woman walking away at some distance from me. The more
attentively I looked at these individuals, the more uneasy I became,
until my terror was completed by the figures slowly turning round
and presenting to me the identical features of you, dear Lilias, and
myself.”
“Maurice, Maurice! you amaze me!”
“Though fully aware of the unearthly nature of these appearances,
I could not resist the desire I felt of following them. I did so, tracing
their silent steps up the glen, until I saw them enter the churchyard
without. I hastened after, but when I too entered the cemetery, the
figures had disappeared!”
The lady’s cheek grew pale as she listened to this narration, for in
those days the belief in such prognostications was universal; and the
time of day when Maurice had seen the wraiths, their retiring
motion, and the fatal spot to which he had traced them, were all
indicative of fast approaching doom. She clung around her husband’s
neck for a few moments in silence, until the deep-seated conviction
of safety while with him, which forms so striking a characteristic of
feminine affection, revived her spirits; and though the tear still hung
on her silken eyelash as she looked up in his face, there was a languid
smile on her cheek as she said,—
“Beshrew you, Maurice, for frightening me so deeply on my
wedding-day! Could you find no other time than this to see bogles?”
“Well said, love,” answered Maurice, who felt no little alarm at
seeing the effect which his story had produced on his wife: “’twas
doubtless a mere delusion.”
“Even should it prove true,” replied Lilias, “we shall at least die
together; and there is a tranquillising influence in that thought,
Maurice, which would go far to make even death agreeable.”
“Let us leave this place,” said Maurice, after the emotion which so
bewitching a confusion excited had in some measure subsided; “I
fear Elspeth will miss us.”
“What then?”
“You know that I have ever distrusted that woman. She and I are
as different from each other as day from darkness. She is a staunch
Covenanter—I a graceless Cavalier. She rails at love-locks, love-
songs, and love-passages—I adore them all. She prays for
MacCallummore, and would fain see his bonnet nod above the crown
of King Charles, and the caps of his merry men;—I would rather see
his head frowning on the Netherbow Port. While she opposed my
suit to you, I only hated her; now that she connives at it—shall I
confess it to you?—I fear her.”
“Nay, now you are unjust. While in the lawful exercise of woman’s
just prerogative,—coquetry,—I seemed to balance the contending
claims of Sir Mungo Campbell and yourself for this poor hand,
Elspeth doubtlessly favoured the cause of her kinsman (all
Campbell’s being of course cousins); but our sovereign will once
unequivocally declared, she became all submission, and has not even
attempted to impugn the decision which we, somewhat foolishly
perhaps, have pronounced in your favour. Besides, Maurice,”
continued Lilias, leaving off the mock-heroic tone in which she had
hitherto spoken for one more akin to natural feeling, “Elspeth
Campbell was my nurse, has a mother’s affection for me, and
therefore would not, I am confident, engage in any scheme inimical
to my happiness.”
“Still she is a Covenanter, and a Campbell,” replied Maurice, “and
as such, her dearest wish, even for your own sake, must be to see you
the wife of him who is both the one and the other.”
“Well,” rejoined Lilias, colouring highly as she spoke, “that at least
you have put out of her power: and yet I regret that I trusted her not
in that matter. It was a secret for a woman, and a nursing mother.”
“Fear not, she shall know in time. I know, I feel it is unmanly, the
dread I entertain; but I cannot quell it. I wish we had not agreed to
make this Logan House the trysting-place of my gallant friends: my
father’s dwelling had been the safer place.”
“Yes; and so have set my worthy guardian, Gillespie Grumach, and
his obsequious friend Sir Mungo, on our track. Come, come, your
alarm is unbecoming. At dawn we leave Logan House. The madcap
disguise which you have prevailed on me to adopt will prevent any
recognition till you have consigned me to my noble kinswoman of
Huntly; and you—but I wrong you—fear not for yourself.”
“Kindly spoken, my love,—would to Heaven you indeed were in
Strathbogie, and I among the gallant Grahams! But here comes
Elspeth, looking as demure as if she were afraid that the idolatrous
sacrifice of the mass, like the leprosy of old, might still stick to those
time-worn walls, and infect her godly heart. Let us go.”
Lilias looked earnestly on the countenance of her nurse as they
met; for though she had not acknowledged so much to Maurice, her
heart had misgiven her as she listened to his discourse. Whether it
might proceed from the melancholy truth, that suspicion once
excited against an individual cannot be entirely quieted by any
innocence whatever, or whether the countenance of Elspeth really
afforded ground for the doubt of her mistress, we are unable to
determine, but certainly the latter imagined at least that she could
detect alarm, solicitude, and fear, lurking amid the apparent
placidity of her nurse’s features.
Nothing was said, however; and the party, remounting their
horses, shortly afterwards arrived at their destination for the night,
namely, the Peel or Tower of Logan House. This edifice, which
crowns the summit of a small knoll or brae on the northern side of
Glencorse water, was one of the many places built for the safety of
the population against any sudden but short-lived attack, and, from
the walls, which are still left, must have been of considerable
strength. It was, at the time we speak of, entire, and consisted of two
storeys; the lower being devoted to the accommodation of the
servants of the house, and that of the family bestial, while the upper
was divided into the few apartments then thought sufficient for the
accommodation of the gentles.
As they rode into the courtyard, Maurice was struck by the want of
attendance which the place betrayed. At that day the laudable
customs of the “queen’s old courtier” had not entirely gone into
desuetude, and every holding, however small, was filled with a
number of retainers, that in the present day would be deemed
excessive. At Logan House, however, things were very different. A
stripling—half-man, half-boy—seemed the only representative of
male vassalage, and the woman-servants, though more numerous,
did not amount to anything near the average number which in those
days divided amongst themselves, with commendable chariness, the
duties of a household.
The faggots, however, blazed cheerfully in the upper apartment,
and food and wine having been prepared in abundance, Maurice for
a moment forgot his suspicions, and Lilias regained her
sprightliness. They conversed gaily together of days gone by, and of
courts and masques and pageants which they had seen, to the
evident discomfort of Elspeth, who not only thought her presence
becoming in her character of nurse, but somewhat necessary in the
existing condition, as she imagined, of the youthful pair. Maurice
soon saw her uneasiness, and wickedly resolved to make it a means
of pastime to himself and Lilias.
“Do you recollect, sweet Lily, when the good King Charles kissed
your cheek in Holyroodhouse, and vowed, on a king’s word, to find a
husband for you?”
“I do; and how a malapert page sounded in my ear that he would
save his Majesty the trouble.”
“And have I not kept my word—ha, lady mine? The great Argyle
and all his men will hardly, I think, undo the links that bind us to
each other;” and inspired, as it seemed, by the pleasant thought, the
youth took the lady’s hand in his, and pressed it warmly and
frequently to his lips.
Elspeth looked on in amazement at the familiarity of intercourse in
which the lady indulged her cousin, and which was equally
repugnant to her natural and acquired feelings on the subject.
“Pshaw! you foolish man, desist!” cried Lilias, blushing and
laughing at the same time, when Maurice attempted to substitute her
rosy lips for the hand he had been so fervently kissing. “What will
Elspeth think?”
“Think, Lady Lilias!” said Elspeth bitterly; “think! I cannot think;
but I can feel for the impropriety—the sinful levity—into which, for
the first time, I see my mistress fallen.”
The fair neck of Lilias crimsoned as she listened to the taunt. For a
moment a frown gathered on her brow, before which the nurse’s
countenance fell; but it died away in a moment, and, with a
beseeching smile, which lay nestled among rosy blushes, she
stretched out her hand and said,—
“Forgive me, Elspeth, we are married!”
This brief annunciation had a striking effect on the individual to
whom it was addressed. She clasped together her withered hands,
and continued for a few moments gazing wildly in the faces of the
startled pair, seemingly anxious to discover there some contradiction
of what she had just heard; and then uttering a loud long shriek,
dashed her face against the wooden board, and groaned audibly.
The terrified Lilias tried to raise the old woman’s head from the
table, but she for some time resisted the kindly effort. At length,
raising her pale and now haggard features to those of the lady, she
exclaimed,—
“Unsay, child of my affection, the dreadful tidings you have told;—
tell me not that I have murdered the daughter of my mistress. Often
when the taish was on me have I seen the dirk in your bosom. Little

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X-Ray Fluorescence in Biological

Sciences: Principles, Instrumentation,

Principles, Instrumentation, and Applications

Vivek Kumar Singh

Durgesh Kumar Tripathi

Limit of Liability/Disclaimer of Warranty

Library of Congress Cataloging-­in-­Publication Data applied for

ISBN: 9781119645542 (Hardback); 9781119645665 (Adobe PDF); 9781119645580 (epub)

Cover Design: Wiley

Set in 9.5/12.5pt STIXTwoText by Straive, Pondicherry, India

List of Contributors xxiii

Part I General Introduction 1

2 X-­Ray Fluorescence for Multi-­elemental Analysis of Vegetation Samples 21

3 X-­Ray Fluorescence Studies of Tea and Coffee 37

6 Use of Energy Dispersive X-­Ray Fluorescence for Clinical Diagnosis 81

7 Preparation of Sample for X-­Ray Fluorescence Analysis 91

Part II Synchrotron Radiation XRF 115

8 Elemental Analysis Using Synchrotron Radiation X-­Ray Fluorescence 117

8.7.6 Micro-­Fluorescence Mapping 142

9 Synchrotron Radiation Based Micro X-­Ray Fluorescence Spectroscopy of Plant

10 Micro X-­Ray Fluorescence Analysis of Toxic Elements in Plants 163

11 Micro X-­Ray Fluorescence Studies of Earthworm (Benthonic Fauna) in Soils and

12 Synchronous Radiation X-­Ray Fluorescence Analysis of Microelements in

Part III Total Reflection XRF 203

13 Total Reflection X-­Ray Fluorescence Analysis of some Biological Samples 205

14 Recent Developments in X-­Ray Fluorescence for Characterization of Nano-­Structured

14.1.3 Calculation of Electric Field Intensity and Fluorescence Intensity 223

15 Total-­Reflection X-­Ray Fluorescence Analysis of Alcoholic and Non-­Alcoholic

Part IV Beginner’s Guide 271

17 Basics and Fundamentals of X-­Rays 273

18 General Principle, Procedures and Detectors of X-­Ray Fluorescence 279

19 Quantitative Analysis in X-­Ray Fluorescence System 287

20 Electronics and Instrumentation for X-­Ray Fluorescence 295

Part V Application to Biological Samples 309

21 Energy Dispersive X-­Ray Fluorescence Analysis of Biological Materials 311

22 X-­Ray Fluorescence Analysis of Milk and Dairy Products 327

23 X-­Ray Fluorescence Analysis of Medicinal Plants 341

23.4 ­ rocedures of Plant Sample Preparation 348

24 X-­Ray Fluorescence Studies of Animal and Human Cell Biology 363

26 X-­Ray Fluorescence for Rapid Detection of Uranium in Blood Extracted from

26.2 ­ hysical Properties of Uranium 387

27 X-­Ray Fluorescence Analysis of Human Hair 405

28 X-­Ray Fluorescence Spectrometry to Study Gallstones, Kidney Stones, Hair, Nails,

28.2.4 XRF Applications to Blood Samples for Trace Detection 428

29 Sampling and Sample Preparation for Chemical Analysis of Plants by Wavelength

30 X-­Ray Fluorescence Analysis in Medical Biology 467

30.2.2.2 XRF Imaging of Metals for Various Diseases 470

31 X-­Ray Fluorescence Analysis in Pharmacology 475

Part VI Special Topics and Comparision with Other Methods 489

32 X-­Ray Fluorescence and State-­of-­the-­Art Related Techniques

33 Lab-scale Wavelength Dispersive X-Ray Fluorescence Spectrometer and Signal

33.1.5.3 Inelastic Scattering 517

34 Chemometric Processing of X-­Ray Fluorescence Data 551

35 X-­Ray Crystallography in Medicinal Biology 563

35.3.6 XRF Use in Recognizing Dental Caries 565

36 Historical Fundamentals of X-­Ray Instruments and Present Trends in Biological

37 X-­Ray Fluorescence Studies of Biological Objects in Mongolia 591

37.3.2 Distribution of Calcium Content in Mongolians’ Hair 599

38 Arsenic Analysis 609

39 X-­Ray Fluorescence: Current Trends and Future Scope 623

39.6 ­ pplications of XRF in the Study of Plant Physiology 629

Tsenddavaa Amartaivan Sandeep Arora

and Department of Metallurgical and Material

Library of Congress Cataloging-in-Publication Data applied for

2 X-Ray Fluorescence for Multi-elemental Analysis of Vegetation Samples 21

3 X-Ray Fluorescence Studies of Tea and Coffee 37

6 Use of Energy Dispersive X-Ray Fluorescence for Clinical Diagnosis 81

7 Preparation of Sample for X-Ray Fluorescence Analysis 91

8 Elemental Analysis Using Synchrotron Radiation X-Ray Fluorescence 117

8.7.6 Micro-Fluorescence Mapping 142

9 Synchrotron Radiation Based Micro X-Ray Fluorescence Spectroscopy of Plant

10 Micro X-Ray Fluorescence Analysis of Toxic Elements in Plants 163

11 Micro X-Ray Fluorescence Studies of Earthworm (Benthonic Fauna) in Soils and

12 Synchronous Radiation X-Ray Fluorescence Analysis of Microelements in

13 Total Reflection X-Ray Fluorescence Analysis of some Biological Samples 205

14 Recent Developments in X-Ray Fluorescence for Characterization of Nano-Structured

15 Total-Reflection X-Ray Fluorescence Analysis of Alcoholic and Non-Alcoholic

17 Basics and Fundamentals of X-Rays 273

18 General Principle, Procedures and Detectors of X-Ray Fluorescence 279

19 Quantitative Analysis in X-Ray Fluorescence System 287

20 Electronics and Instrumentation for X-Ray Fluorescence 295

21 Energy Dispersive X-Ray Fluorescence Analysis of Biological Materials 311

22 X-Ray Fluorescence Analysis of Milk and Dairy Products 327

23 X-Ray Fluorescence Analysis of Medicinal Plants 341

23.4 rocedures of Plant Sample Preparation 348

24 X-Ray Fluorescence Studies of Animal and Human Cell Biology 363

26 X-Ray Fluorescence for Rapid Detection of Uranium in Blood Extracted from

26.2 hysical Properties of Uranium 387

27 X-Ray Fluorescence Analysis of Human Hair 405

28 X-Ray Fluorescence Spectrometry to Study Gallstones, Kidney Stones, Hair, Nails,

30 X-Ray Fluorescence Analysis in Medical Biology 467

31 X-Ray Fluorescence Analysis in Pharmacology 475

32 X-Ray Fluorescence and State-of-the-Art Related Techniques

34 Chemometric Processing of X-Ray Fluorescence Data 551

35 X-Ray Crystallography in Medicinal Biology 563

36 Historical Fundamentals of X-Ray Instruments and Present Trends in Biological

37 X-Ray Fluorescence Studies of Biological Objects in Mongolia 591

39 X-Ray Fluorescence: Current Trends and Future Scope 623

39.6 pplications of XRF in the Study of Plant Physiology 629

December, 2021 Dr. Vivek Kumar Singh

1.2 Analytical Capabilities of XRF and Micro-XRF

1.3 Comparison with Other Analytical Methods

1.3.2.1 Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

1.3.3 Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)

1.3.5 Laser-Induced Breakdown Spectroscopy (LIBS)

Figure 1.3 A schematic setup of laser-induced breakdown spectroscopy (LIBS).

1.3.6 Proton-Induced X-Ray Emission (PIXE)

Table 1.3 Advantages and limitations of SEM-EDS method [3, 8].

1.3.7 Scanning Electron Microscopy–Energy Dispersive X–Ray Spectroscopy (SEM-EDS)

1.3.7.1 Differences in XRF and SEM-EDS (Sample Handling, Experimental Conditions,

Figure 1.4 K-shell cross sections for 10,000

X-denotes the emission due to X-rays.