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NISTory of the Periodic Table
Sodium:
NIST scientists used
lasers to cool a gas of Rubidium:
these atoms to more than
Cesium: theoretically expected to These atoms were used by
temperatures even closer researchers at JILA (NIST-CU Boulder) Potassium and
The frequency of microwave
owave to absolute zero.
1988 to create the first Bose-Einstein Rubidium:
radiation from this atom
m in (Nobel Prize 1997) condensate (Nobel Prize 2001).
atomic clocks such as the Image Credit: H.Mark Helfer/NI
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define the second. ultracold gas of molecules
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1967 1995 and demonstrated striking
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was concentrated at atoms were probed in a
NIST and then identified NIST trap to create
by Columbia University's "quantum logic" clocks that
Harold Urey (Nobel Prize 1934). On the measured the second more precisely than
left is a deuterium lamp; the light on the before and tested Einstein's general theory
right comes from the NIST SURF III of relativity. Such quantum manipulations
Synchrotron Ultraviolet Radiation Facility. were recognized in the 2012 Nobel Prize.
Image Credit: Uwe Arp/NIST Image Credit: J. Amini/NIST
www.nist.gov Design by: Natasha Hanacek/NIST

Undergraduate Instrumental Analysis
Analytical instrumentation is crucial to research in molecular biology, medicine, geology, food science, materials
science, forensics, and many other fields. Undergraduate Instrumental Analysis, 8th Edition, provides the reader with an
understanding of all major instrumental analyses, and is unique in that it starts with the fundamental principles, and then
develops the level of sophistication that is needed to make each method a workable tool for the student. Each chapter
includes a discussion of the fundamental principles underlying each technique, detailed descriptions of the instrumenta-
tion, and a large number of applications. Each chapter includes an updated bibliography and problems, and most chapters
have suggested experiments appropriate to the technique.
This edition has been completely updated, revised, and expanded. The order of presentation has been changed from the 7th
edition in that after the introduction to spectroscopy, UV-Vis is discussed. This order is more in keeping with the prefer-
ence of most instructors. Naturally, once the fundamentals are introduced, instructors are free to change the order of pres-
entation. Mathematics beyond algebra is kept to a minimum, but for the interested student, in this edition we provide an
expanded discussion of measurement uncertainty that uses elementary calculus (although a formula approach can be used
with no loss of context). Unique among all instrumental analysis texts we explicitly discuss safety, up front in Chapter 2.
The presentation intentionally avoids a finger-wagging, thou-shalt-not approach in favor of a how-to discussion of good
laboratory and industrial practice. It is focused on hazards (and remedies) that might be encountered in the use of instru-
mentation. Among the new topics introduced in this edition are:
• Photoacoustic spectroscopy.
• Cryogenic NMR probes and actively shielded magnets.
• The nature of mixtures (in the context of separations).
• Troubleshooting and leaks in high vacuum systems such as mass spectrometers.
• Instrumentation laboratory safety.
• Standard reference materials and standard reference data.
In addition, the authors have included many instrument manufacturer’s websites, which contain extensive resources.
We have also included many government websites and a discussion of resources available from National Measurement
Laboratories in all industrialized countries. Students are introduced to standard methods and protocols developed by regu-
latory agencies and consensus standards organizations in this context as well.
Caption for cover art:
Photographs: L: Prof. April Hill of Metropolitan State University of Denver instructs a class in analytical instrumentation
(courtesy of Metropolitan State University of Denver); R: Prof Lisa Ott of California State University, Chico, works with a
research student in the instrumentation laboratory (courtesy of California State University, Chico).
Spectra, clockwise from top: X-ray photoelectron spectrum of lunar soil; mass spectrum of ultralow sulfur diesel fuel;
infrared spectrum of rocket propellant 1; gas chromatogram of aviation kerosene; fully coupled NMR spectrum of sucrose
in D2O; hypothetical TGA and DTA curves.
Rights information:
I have permission from Tim Carrol of Metro State in Denver for the April Hill picture, given by email; I have permission
from Jason Halley of California State University, Chico for the Lisa Ott picture, given on a standard form. Both are aware
that it is for cover art. The X-ray spectrum is from the book, the MS is from my own work, the IR is from my own work,
the chromatogram is from my own work, the NMR is from the book, the TA/DTA is from the book.
Undergraduate Instrumental Analysis
Eighth Edition
Thomas J. Bruno, James W. Robinson,

Eileen M. Skelly Frame, and George M. Frame II
Eighth edition published 2024
by CRC Press
2385 NW Executive Center Drive, Suite 320, Boca Raton FL 33431
and by CRC Press
4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
CRC Press is an imprint of Taylor & Francis Group, LLC
© 2024 Taylor & Francis Group, LLC
First edition published by Routledge 1970
Seventh edition published by Routledge 2014
Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity
of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in
this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been
acknowledged please write and let us know so we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,
mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or
retrieval system, without written permission from the publishers.
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Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without
intent to infringe.
Library of Congress Cataloging‑in‑Publication Data
Names: Bruno, Thomas J., author. | Robinson, James W., 1923–2018, author. | Frame, George M., II, author. |
Frame, Eileen M. Skelly, 1953– author.
Title: Undergraduate instrumental analysis / Thomas J. Bruno, James W. Robinson, Eileen M. Skelly Frame,
and Dr. George M. Frame II.
Description: Eighth edition. | Boca Raton, FL : CRC Press, 2023. | Revised edition of: Undergraduate instrumental analysis /
James W. Robinson, Eileen Skelly Frame, George M. Frame II. Seventh edition. 2014. |
Includes bibliographical references and index. |
Identifiers: LCCN 2022041820 | ISBN 9781032036915 (hbk) | ISBN 9781032036953 (pbk) | ISBN 9781003188544 (ebk)
Subjects: LCSH: Instrumental analysis–Textbooks.
Classification: LCC QD79.I5 R6 2023 | DDC 543–dc23/eng20230111
LC record available at https://lccn.loc.gov/2022041820
ISBN: 9781032036915 (hbk)
ISBN: 9781032036953 (pbk)
ISBN: 9781003188544 (ebk)
DOI: 10.1201/9781003188544
Typeset in Times
by Newgen Publishing UK
Access the Support Material: www.routledge.com/9781032036953
Contents
Preface ��xxiii
Acknowledgments ��xxvii
Authors��xxix
Chapter 1
Concepts of Instrumental Analytical Chemistry�� 1
1.1 Introduction: What Is Analytical Chemistry?............................................................................................................ 1
1.1.1 The Difference between Weight and Mass.................................................................................................. 2
1.2 Analytical Approach.................................................................................................................................................. 3
1.2.1 Defining the Problem................................................................................................................................... 3
1.2.1.1 Qualitative Analysis.................................................................................................................... 4
1.2.1.2 Quantitative Analysis.................................................................................................................. 5
1.2.2 Designing the Analytical Method.............................................................................................................. 10
1.2.3 Sampling.................................................................................................................................................... 11
1.2.3.1 Gas Samples.............................................................................................................................. 13
1.2.3.2 Liquid Samples......................................................................................................................... 14
1.2.3.3 Solid Samples...........................................................................................................................14
1.2.4 Storage of Samples.................................................................................................................................... 14
1.3 A Conceptual Discussion about Uncertainty in Measurements.............................................................................. 15
1.4 Basic Statistics and Data Handling.......................................................................................................................... 17
1.4.1 Significant Figures..................................................................................................................................... 17
1.4.2 Accuracy and Precision............................................................................................................................. 18
1.4.3 Types of Errors........................................................................................................................................... 19
1.4.3.1 Determinate Error..................................................................................................................... 20
1.4.3.2 Indeterminate Error................................................................................................................... 22
1.4.4 Definitions for Statistics............................................................................................................................ 23
1.4.5 Quantifying Random Error........................................................................................................................ 23
1.4.5.1 Confidence Limits..................................................................................................................... 26
1.4.5.2 Variance..................................................................................................................................... 27
1.4.6 Detection of Outliers.................................................................................................................................. 27
1.4.7 Expressing Uncertainty.............................................................................................................................. 28
1.5 Sample Preparation.................................................................................................................................................. 29
1.5.1 Acid Dissolution and Digestion................................................................................................................. 29
1.5.2 Fusions....................................................................................................................................................... 31
1.5.3 Dry Ashing and Combustion.....................................................................................................................31
1.5.4 Extraction................................................................................................................................................... 32
1.5.4.1 Solvent Extraction..................................................................................................................... 32
1.5.4.2 Solid-Phase Extraction.............................................................................................................. 35
1.5.4.3 QuEChERS............................................................................................................................... 35
1.5.4.4 Solid-Phase Microextraction..................................................................................................... 36
1.6 Performing the Measurement.................................................................................................................................. 38
1.6.1 Signals and Noise...................................................................................................................................... 39
1.6.2 Plotting Calibration Curves....................................................................................................................... 41
1.7 Assessing the Data................................................................................................................................................... 42
1.7.1 Limit of Detection...................................................................................................................................... 43
1.7.2 Limit of Quantitation................................................................................................................................. 43
1.8 Reference Materials and Reference Data................................................................................................................ 44
1.8.1 Reference Materials................................................................................................................................... 44
1.8.2 Reference Data........................................................................................................................................... 44
Problems............................................................................................................................................................................... 45
Bibliography......................................................................................................................................................................... 46
vii
viiiContents
Chapter 2
Safety in the Instrumentation Laboratory�� 49
2.1 Electrical Hazards and Safety.................................................................................................................................. 50
2.1.1 Electrical Lines, Receptacles, and Cables................................................................................................. 50
2.1.2 PAT Testing Procedures in the U.K........................................................................................................... 55
2.1.3 Electrical Shocks....................................................................................................................................... 55
2.1.4 Batteries..................................................................................................................................................... 55
2.1.5 Portable Generators................................................................................................................................... 56
2.1.6 A Brief Discussion about Ground.............................................................................................................. 57
2.2 Instrument Cooling.................................................................................................................................................. 58
2.3 Compressed Gas Cylinder Safety............................................................................................................................ 58
2.3.1 Compressed Gas Regulators and Manifolds.............................................................................................. 59
2.3.2 Gas Cylinder Markings.............................................................................................................................. 60
2.4 Cryogenic Fluid Safety............................................................................................................................................ 61
2.4.1 Cryogenic Fluids........................................................................................................................................ 61
2.4.2 Cryogenic Liquid Containers..................................................................................................................... 63
2.5 The Use of Fume Hoods in the Instrumentation Laboratory................................................................................... 64
2.6 Laser Safety............................................................................................................................................................. 66
2.7 Compressed Air Line Safety.................................................................................................................................... 67
2.8 Ladder Safety........................................................................................................................................................... 67
2.8.1 Step Ladders.............................................................................................................................................. 67
2.8.2 Straight, Combination, and Extension Ladders......................................................................................... 68
2.9 Hearing Protection................................................................................................................................................... 68
2.10 Selection of Laboratory Gloves............................................................................................................................... 69
2.11 Selection of Respirator Cartridges and Filters......................................................................................................... 69
2.12 Safety with Nanomaterials....................................................................................................................................... 69
2.13 Relative Dose Ranges from Ionizing Radiation......................................................................................................73
2.14 Magnetic Field Safety.............................................................................................................................................. 75
2.15 Miscellaneous Hazardous Conditions and Fire Safety............................................................................................ 76
2.15.1 Fire Extinguishers...................................................................................................................................... 77
2.15.2 Hot Work Permits...................................................................................................................................... 78
2.16 Stop Work Orders.................................................................................................................................................... 78
References............................................................................................................................................................................. 80
Chapter 3
Introduction to Spectroscopy�� 81
3.1 Interaction between Electromagnetic Radiation and Matter................................................................................... 81
3.1.1 What Is Electromagnetic Radiation?......................................................................................................... 81
3.1.2 How Does Electromagnetic Radiation Interact with Matter?.................................................................... 82
3.2 Atoms and Atomic Spectroscopy............................................................................................................................. 86
3.3 Molecules and Molecular Spectroscopy.................................................................................................................. 87
3.3.1 Rotational Transitions in Molecules.......................................................................................................... 87
3.3.2 Vibrational Transitions in Molecules......................................................................................................... 88
3.3.3 Electronic Transitions in Molecules.......................................................................................................... 88
3.4 Absorption Laws...................................................................................................................................................... 89
3.4.1 Deviations from Beer’s Law...................................................................................................................... 91
3.5 Methods of Calibration............................................................................................................................................ 92
3.5.1 Calibration with Standards........................................................................................................................ 92
3.5.2 Method of Standard Additions................................................................................................................... 93
3.5.3 Internal Standard Calibration..................................................................................................................... 96
3.5.4 Errors Associated with Beer’s Law Relationships..................................................................................... 98
3.6 Optical Systems Used in Spectroscopy................................................................................................................. 100
3.6.1 Radiation Sources.................................................................................................................................... 101
3.6.2 Wavelength Selection Devices................................................................................................................. 102
Contents ix
3.6.2.1 Filters...................................................................................................................................... 102

3.6.2.2 Monochromator....................................................................................................................... 102
3.6.2.3 Resolution Required to Separate Two Lines of Different Wavelengths.................................. 104
3.6.3 Optical Slits............................................................................................................................................. 108
3.6.4 Detectors.................................................................................................................................................. 110
3.6.5 Single-Beam and Double-Beam Optics................................................................................................... 110
3.6.6 Dispersive Optical Layouts...................................................................................................................... 112
3.6.7 Fourier Transform Spectrometers............................................................................................................ 113
3.6.7.1 Advantages of FT Systems..................................................................................................... 114
3.7 Spectroscopic Technique and Instrument Nomenclature....................................................................................... 115
Suggested Experiments....................................................................................................................................................... 115
Problems............................................................................................................................................................................. 116
Bibliography....................................................................................................................................................................... 118
Chapter 4
Visible and Ultraviolet Molecular Spectroscopy�� 119
4.1 Introduction............................................................................................................................................................ 119
4.1.1 Electronic Excitation in Molecules.......................................................................................................... 122
4.1.2 Absorption by Molecules......................................................................................................................... 124
4.1.3 Molar Absorptivity................................................................................................................................... 125
4.1.4 Shape of UV Absorption Curves............................................................................................................. 125
4.1.5 Solvents for UV/VIS Spectroscopy......................................................................................................... 128
4.2 Instrumentation...................................................................................................................................................... 129
4.2.1 Optical System......................................................................................................................................... 129
4.2.3 Monochromators...................................................................................................................................... 132
4.2.4 Detectors.................................................................................................................................................. 132
4.2.4.1 Barrier Layer Cell................................................................................................................... 133
4.2.4.2 Photomultiplier Tube.............................................................................................................. 134
4.2.4.3 Semiconductor Detectors: Diodes and Diode Array Systems................................................. 135
4.2.4.4 Diodes..................................................................................................................................... 136
4.2.4.5 Diode Arrays........................................................................................................................... 137
4.2.5 Sample Holders........................................................................................................................................ 138
4.2.5.1 Liquid and Gas Cells............................................................................................................... 138
4.2.5.2 Matched Cells......................................................................................................................... 139
4.2.5.3 Flow-Through Samplers......................................................................................................... 140
4.2.5.4 Solid Sample Holders............................................................................................................. 140
4.2.5.5 Fiber-Optic Probes.................................................................................................................. 141
4.2.6 Microvolume, Nanovolume, and Handheld UV/VIS Spectrometers....................................................... 141
4.2.6.1 Microvolume Systems............................................................................................................. 142
4.2.6.2 Variable Path Length Slope Spectroscopy™ System.............................................................143
4.2.6.3 Handheld Visible Spectroscopy System.................................................................................. 147
4.3 UV Absorption Spectra of Molecules.................................................................................................................... 150
4.3.1 Solvent Effects on UV Spectra................................................................................................................ 150
4.3.1.1 Bathochromic or Red Shift..................................................................................................... 150
4.3.1.2 Hypsochromic or Blue Shift................................................................................................... 151
4.4 UV Spectra and the Structure of Organic Molecules............................................................................................ 152
4.4.1 Conjugated Diene Systems...................................................................................................................... 152
4.4.2 Conjugated Ketone Systems.................................................................................................................... 154
4.4.3 Substitution of Benzene Rings................................................................................................................. 156
4.5 Analytical Applications......................................................................................................................................... 156
4.5.1 Qualitative Structural Analysis................................................................................................................ 156
4.5.2 Quantitative Analysis............................................................................................................................... 157
4.5.3 Multicomponent Determinations............................................................................................................. 162
4.5.4 Other Applications................................................................................................................................... 162
xContents
4.5.4.1 Reaction Kinetics.................................................................................................................... 162

4.5.4.2 Spectrophotometric Titrations................................................................................................. 163
4.5.4.3 Spectroelectrochemistry.......................................................................................................... 163
4.5.4.4 Analysis of Solids................................................................................................................... 164
4.5.5 Measurement of Color............................................................................................................................. 164
4.6 Accuracy and Precision in UV/VIS Absorption Spectrometry.............................................................................. 166
4.7 Nephelometry and Turbidimetry............................................................................................................................ 166
4.8 Molecular Emission Spectrometry........................................................................................................................ 167
4.8.1 Fluorescence and Phosphorescence......................................................................................................... 167
4.8.2 Relationship between Fluorescence Intensity and Concentration........................................................... 169
4.9 Instrumentation for Luminescence Measurements................................................................................................ 170
4.9.1 Wavelength Selection Devices................................................................................................................. 170
4.9.3 Detectors.................................................................................................................................................. 172
4.9.4 Sample Cells............................................................................................................................................ 173
4.9.5 Spectrometer Systems.............................................................................................................................. 173
4.10 Analytical Applications of Luminescence............................................................................................................. 173
4.10.1 Advantages of Fluorescence and Phosphorescence................................................................................. 175
4.10.2 Disadvantages of Fluorescence and Phosphorescence............................................................................ 175
Suggested Experiments.......................................................................................................................................... 176
Problems................................................................................................................................................................. 177
Bibliography........................................................................................................................................................... 180
Chapter 5
Infrared, Near-Infrared, Raman, and Photoacoustic Spectroscopy�� 183
5.1 Absorption of IR Radiation by Molecules............................................................................................................. 184
5.1.1 Dipole Moments in Molecules................................................................................................................. 184
5.1.2 Types of Vibrations in Molecules............................................................................................................ 185
5.1.3 Vibrational Motion................................................................................................................................... 187
5.2 IR Instrumentation................................................................................................................................................. 190
5.2.1.1 Mid-IR Sources....................................................................................................................... 191
5.2.1.2 NIR Sources............................................................................................................................ 193
5.2.1.3 Far-IR Sources........................................................................................................................ 193
5.2.1.4 IR Laser Sources..................................................................................................................... 193
5.2.2 Monochromators and Interferometers..................................................................................................... 194
5.2.2.1 FT Spectrometers.................................................................................................................... 194
5.2.2.2 Interferometer Components.................................................................................................... 197
5.2.3 Detectors.................................................................................................................................................. 198
5.2.3.1 Bolometer................................................................................................................................ 198
5.2.3.2 Thermocouples........................................................................................................................ 199
5.2.3.3 Thermistors............................................................................................................................. 199
5.2.3.4 Golay Detector........................................................................................................................ 199
5.2.3.5 Pyroelectric Detectors............................................................................................................. 199
5.2.3.6 Photon Detectors..................................................................................................................... 200
5.2.4 Detector Response Time.......................................................................................................................... 200
5.3 Sampling Techniques............................................................................................................................................. 201
5.3.1 Techniques for Transmission (Absorption) Measurements..................................................................... 201
5.3.1.1 Solid Samples.........................................................................................................................201
5.3.1.2 Liquid Samples....................................................................................................................... 202
5.3.1.3 Gas Samples............................................................................................................................ 204
5.3.2 Background Correction in Transmission Measurements......................................................................... 205
5.3.2.1 Solvent Absorption.................................................................................................................. 205
5.3.2.2 Air Absorption........................................................................................................................ 205
5.3.3 Techniques for Reflectance and Emission Measurements....................................................................... 206
Contents xi
5.3.3.1 Attenuated Total Reflectance.................................................................................................. 206

5.3.3.2 Specular Reflectance............................................................................................................... 208
5.3.3.3 Diffuse Reflectance................................................................................................................. 209
5.3.3.4 IR Emission............................................................................................................................. 209
5.4 FTIR Microscopy................................................................................................................................................... 210
5.5 Nondispersive IR Systems..................................................................................................................................... 214
5.6 Analytical Applications of IR Spectroscopy.......................................................................................................... 214
5.6.1 Qualitative Analyses and Structural Determination by Mid-IR Absorption Spectroscopy..................... 215
5.6.1.1 Hydrocarbons.......................................................................................................................... 218
5.6.1.2 Organic Compounds with C–O Bonds...................................................................................222
5.6.1.3 Nitrogen-Containing Organic Compounds............................................................................. 227
5.6.1.4 Functional Groups Containing Heteroatoms.......................................................................... 228
5.6.2 Quantitative Analyses by IR Spectrometry.............................................................................................. 233
5.7 NIR Spectroscopy.................................................................................................................................................. 236
5.7.1 Instrumentation........................................................................................................................................ 236
5.7.2 NIR Vibrational Bands, Spectral Interpretation, and Calibration............................................................ 236
5.7.2.1 NIR Calibration: Chemometrics............................................................................................. 238
5.7.3 Sampling Techniques for NIR Spectroscopy........................................................................................... 238
5.7.3.1 Liquids and Solutions............................................................................................................. 239
5.7.3.2 Solids......................................................................................................................................240
5.7.3.3 Gases....................................................................................................................................... 240
5.7.4 Applications of NIR Spectroscopy.......................................................................................................... 240
5.8 Raman Spectroscopy............................................................................................................................................. 242
5.8.1 Principles of Raman Scattering............................................................................................................... 242
5.8.2 Raman Instrumentation............................................................................................................................ 244
5.8.2.1 Light Sources.......................................................................................................................... 244
5.8.2.2 Dispersive Spectrometer Systems........................................................................................... 245
5.8.2.3 FT-Raman Spectrometers........................................................................................................ 246
5.8.2.4 Fiber-Optic-Based Modular and Handheld Systems.............................................................. 246
5.8.2.5 Samples and Sample Holders for Raman Spectroscopy......................................................... 249
5.8.3 Applications of Raman Spectroscopy...................................................................................................... 249
5.8.4 Resonance Raman Effect......................................................................................................................... 254
5.8.5 Surface-Enhanced Raman Spectroscopy................................................................................................. 254
5.8.6 Raman Microscopy.................................................................................................................................. 255
5.9 Chemical Imaging Using NIR, IR, and Raman Spectroscopy............................................................................... 256
5.10 Photoacoustic Spectroscopy.................................................................................................................................. 262
Inset: Alexander Graham Bell............................................................................................................................................. 265
Problems............................................................................................................................................................................. 268
Bibliography....................................................................................................................................................................... 273
Spectral Databases.............................................................................................................................................................. 275
Chapter 6
Magnetic Resonance Spectroscopy�� 277
6.1 Introduction to Nuclear Magnetic Resonance Spectroscopy................................................................................. 277
6.1.1 Properties of Nuclei................................................................................................................................. 278
6.1.2 Quantization of 1H Nuclei in a Magnetic Field....................................................................................... 279
6.1.2.1 Saturation and Magnetic Field Strength................................................................................. 281
6.1.3 Width of Absorption Lines......................................................................................................................284
6.1.3.1 Homogeneous Field................................................................................................................ 284
6.1.3.2 Relaxation Time...................................................................................................................... 284
6.1.3.3 The Chemical Shift................................................................................................................. 285
6.1.3.4 Magic Angle Spinning............................................................................................................ 285
6.1.3.5 Other Sources of Line Broadening......................................................................................... 286
6.2 FT-NMR Experiment............................................................................................................................................. 287
xiiContents
6.3 Chemical Shifts...................................................................................................................................................... 287

6.4 Spin–Spin Coupling............................................................................................................................................... 291
6.5.1 Sample Holder......................................................................................................................................... 302
6.5.2 Sample Probe........................................................................................................................................... 305
6.5.2.1 Cryogenically Cooled Probes................................................................................................. 305
6.5.3 Magnet..................................................................................................................................................... 305
6.5.4 RF Generation and Detection.................................................................................................................. 308
6.5.5 Signal Integrator and Computer............................................................................................................... 309
6.6 Analytical Applications of NMR........................................................................................................................... 309
6.6.1 Samples and Sample Preparation for NMR............................................................................................. 309
6.6.2 Qualitative Analyses: Molecular Structure Determination...................................................................... 310
6.6.2.1 Relationship between the Area of a Peak and Molecular Structure........................................ 310
6.6.2.2 Chemical Exchange................................................................................................................ 311
6.6.2.3 Double-Resonance Experiments............................................................................................. 312
6.6.3 Interpretation of Proton Spectra............................................................................................................... 313
6.6.3.1 Aliphatic Alkanes, Alkenes, Alkynes, and Alkyl Halides....................................................... 315
6.6.3.2 Aromatic Compounds............................................................................................................. 319
6.6.3.3 Oxygen-Containing Organic Compounds............................................................................... 321
6.6.3.4 Nitrogen-Containing Organic Compounds............................................................................. 321
6.6.4 13
C NMR.................................................................................................................................................. 324
6.6.4.1 Heteronuclear Decoupling...................................................................................................... 326
6.6.4.2 Nuclear Overhauser Effect...................................................................................................... 326
6.6.4.3 13C NMR Spectra of Solids..................................................................................................... 328
6.6.4.4 Interpretation of 13C Spectra................................................................................................... 330
6.6.5 2D NMR.................................................................................................................................................. 332
6.6.6 Qualitative Analyses: Other Applications................................................................................................ 335
6.6.7 Quantitative Analyses.............................................................................................................................. 338
6.7 Hyphenated NMR Techniques............................................................................................................................... 342
6.8 NMR Imaging and MRI......................................................................................................................................... 343
6.9 Time Domain NMR............................................................................................................................................... 347
6.9.1 Solid Fat Content Determination by TD-NMR....................................................................................... 348
6.9.2 Field Homogeneity and Spin Echo.......................................................................................................... 349
6.9.3 Relaxation Time Distribution..................................................................................................................351
6.9.4 TD-NMR Applications............................................................................................................................ 352
6.10 Low-Field, Portable, and Miniature NMR Instruments......................................................................................... 353
6.11 Limitations of NMR.............................................................................................................................................. 353
6.12 Electron Spin Resonance Spectroscopy................................................................................................................. 353
6.12.1.1 Samples and Sample Holders.................................................................................................. 357
6.12.2 Miniature ESR Spectroscopy................................................................................................................... 358
6.12.3 ESR Spectra and Hyperfine Interactions................................................................................................. 360
6.12.4 Applications............................................................................................................................................. 361
6.12.4.1 Spin Labels and Spin Traps....................................................................................................363
6.12.5 CW-ENDOR............................................................................................................................................ 364
Problems............................................................................................................................................................................. 366
Bibliography....................................................................................................................................................................... 372
Spectral Databases.............................................................................................................................................................. 373
Chapter 7
Atomic Absorption Spectrometry�� 375
7.1 Absorption of Radiant Energy by Atoms............................................................................................................... 375
7.1.1 Spectral Linewidth................................................................................................................................... 377
7.1.2 Degree of Radiant Energy Absorption..................................................................................................... 377
Contents xiii

7.2.1.1 Hollow Cathode Lamp............................................................................................................ 378
7.2.1.2 Electrodeless Discharge Lamp................................................................................................ 379
7.2.2 Atomizers................................................................................................................................................. 380
7.2.2.1 Flame Atomizers..................................................................................................................... 380
7.2.2.2 Electrothermal Atomizers....................................................................................................... 382
7.2.2.3 Other Atomizers...................................................................................................................... 384
7.2.3 Spectrometer Optics................................................................................................................................. 384
7.2.3.1 Monochromator....................................................................................................................... 384
7.2.3.2 Optics and Spectrometer Configuration.................................................................................. 384
7.2.4 Detectors.................................................................................................................................................. 386
7.2.5 Modulation............................................................................................................................................... 386
7.2.6 Commercial AAS Systems...................................................................................................................... 386
7.2.6.1 High-Resolution Continuum Source AAS.............................................................................. 387
7.3 Atomization Process.............................................................................................................................................. 387
7.3.1 Flame Atomization................................................................................................................................... 387
7.3.2 Graphite Furnace Atomization................................................................................................................. 391
7.4 Interferences in AAS.............................................................................................................................................. 392
7.4.1 Nonspectral Interferences........................................................................................................................ 392
7.4.1.1 Chemical Interference............................................................................................................. 392
7.4.1.2 Matrix Interference................................................................................................................. 393
7.4.1.3 Ionization Interference............................................................................................................ 394
7.4.1.4 Nonspectral Interferences in GFAAS..................................................................................... 394
7.4.1.5 Chemical Modification............................................................................................................ 395
7.4.2 Spectral Interferences.............................................................................................................................. 397
7.4.2.1 Atomic Spectral Interference.................................................................................................. 397
7.4.2.2 Background Absorption and Its Correction............................................................................ 397
7.4.2.3 Two-Line Background Correction.......................................................................................... 397
7.4.2.4 Continuum Source Background Correction............................................................................ 397
7.4.2.5 Zeeman Background Correction............................................................................................. 399
7.4.2.6 Smith–Hieftje Background Corrector..................................................................................... 400
7.4.2.7 Spectral Interferences in GFAAS............................................................................................ 401
7.5 Analytical Applications of AAS............................................................................................................................ 402
7.5.1 Qualitative Analysis................................................................................................................................. 402
7.5.2 Quantitative Analysis............................................................................................................................... 402
7.5.2.1 Quantitative Analytical Range................................................................................................ 402
7.5.2.2 Calibration............................................................................................................................... 403
7.5.3 Analysis of Samples................................................................................................................................ 404
7.5.3.1 Liquid Samples....................................................................................................................... 404
7.5.3.2 Solid Samples.........................................................................................................................405
7.5.3.3 Gas Samples............................................................................................................................ 407
7.5.3.4 Cold Vapor Mercury Technique.............................................................................................. 407
7.5.3.5 Hydride Generation Technique............................................................................................... 408
7.5.3.6 Flow Injection Analysis.......................................................................................................... 408
7.5.3.7 Flame Microsampling............................................................................................................. 409
Problems............................................................................................................................................................................. 410
7.A Appendix...................................................................................................................................................................... 411
7.B Appendix...................................................................................................................................................................... 415
Bibliography....................................................................................................................................................................... 418
Chapter 8
Atomic Emission Spectroscopy�� 421
8.1 Flame Atomic Emission Spectroscopy.................................................................................................................. 421
xivContents
8.1.1 Instrumentation for Flame OES............................................................................................................... 422

8.1.1.1 Burner Assembly..................................................................................................................... 422
8.1.1.2 Wavelength Selection Devices................................................................................................ 423
8.1.1.3 Detectors................................................................................................................................. 424
8.1.1.4 Flame Excitation Source......................................................................................................... 424
8.1.2 Interferences............................................................................................................................................ 425
8.1.2.2 Excitation and Ionization Interferences.................................................................................. 426
8.1.2.3 Spectral Interferences.............................................................................................................. 426
8.1.3 Analytical Applications of Flame OES.................................................................................................... 427
8.1.3.1 Qualitative Analysis................................................................................................................ 427
8.1.3.2 Quantitative Analysis.............................................................................................................. 427
8.2 Atomic OES........................................................................................................................................................... 429
8.2.1 Instrumentation for Emission Spectroscopy............................................................................................ 430
8.2.1.1 Electrical Excitation Sources.................................................................................................. 431
8.2.1.2 Sample Holders....................................................................................................................... 435
8.2.1.3 Spectrometers.......................................................................................................................... 435
8.2.1.4 Detectors................................................................................................................................. 437
8.2.2 Interferences in Arc and Spark Emission Spectroscopy.......................................................................... 441
8.2.2.1 Matrix Effects and Sample Preparation.................................................................................. 441
8.2.2.2 Spectral Interference............................................................................................................... 442
8.2.2.3 Internal Standard Calibration.................................................................................................. 442
8.2.3 Applications of Arc and Spark Emission Spectroscopy.......................................................................... 443
8.2.3.1 Qualitative Analysis................................................................................................................ 443
8.2.3.2 Raies Ultimes.......................................................................................................................... 443
8.3 Plasma Emission Spectroscopy............................................................................................................................. 446
8.3.1 Instrumentation for Plasma Emission Spectrometry............................................................................... 446
8.3.1.1 Excitation Sources.................................................................................................................. 446
8.3.1.2 Spectrometer Systems for ICP, DCP, and MP......................................................................... 451
8.3.1.3 Sample Introduction Systems................................................................................................. 453
8.3.2 Interferences and Calibration in Plasma Emission Spectrometry............................................................ 458
8.3.2.1 Chemical and Ionization Interference..................................................................................... 459
8.3.2.2 Spectral Interference and Correction...................................................................................... 459
8.3.3 Applications of ICP, DCP, and MP Atomic Emission Spectroscopy....................................................... 462
8.3.4 Chemical Speciation with Hyphenated Instruments................................................................................ 464
8.4 GD Emission Spectrometry................................................................................................................................... 465
8.4.1 DC and RF GD Sources........................................................................................................................... 465
8.4.2 Applications of GD Atomic Emission Spectrometry............................................................................... 466
8.4.2.1 Bulk Analysis.......................................................................................................................... 466
8.4.2.2 Depth Profile Analysis............................................................................................................ 466
8.5 Atomic Fluorescence Spectroscopy....................................................................................................................... 467
8.5.1 Instrumentation for AFS.......................................................................................................................... 469
8.5.2 Interferences in AFS................................................................................................................................ 470
8.5.2.2 Spectral Interference............................................................................................................... 470
8.5.3 Applications of AFS................................................................................................................................ 470
8.5.3.1 Graphite Furnace Laser-Excited AFS..................................................................................... 471
8.5.3.2 Mercury Determination and Speciation by AFS..................................................................... 471
8.5.3.3 Hydride Generation and Speciation by AFS........................................................................... 472
8.6 Commercial Atomic Emission Systems................................................................................................................. 472
8.6.1 Arc and Spark Systems............................................................................................................................ 472
8.6.2 ICP, DCP, and MP Systems..................................................................................................................... 472
8.6.3 GD Systems............................................................................................................................................. 473
8.6.4 AFS Systems............................................................................................................................................ 473
Contents xv
8.7 Laser-Induced Breakdown Spectroscopy.............................................................................................................. 473

8.7.1 Principle of Operation.............................................................................................................................. 473
8.7.3 Applications of LIBS............................................................................................................................... 475
8.7.3.1 Qualitative Analysis and Semiquantitative Analysis............................................................... 476
8.7.3.3 Remote Analysis..................................................................................................................... 477
8.7.4 Commercial LIBS Systems...................................................................................................................... 480
8.8 Atomic Emission Literature and Resources.......................................................................................................... 480
8.9 Comparison of Atomic Spectroscopic and ICP-MS Techniques........................................................................... 480
Problems............................................................................................................................................................................. 481
8.A Appendix...................................................................................................................................................................... 483
8.B Appendix: Comparison of Atomic Spectroscopic Analytical Techniques.................................................................... 484
Bibliography....................................................................................................................................................................... 486
Chapter 9
X-Ray Spectroscopy........................................................................................................................................................... 489
Contributing authors: Alexander Seyfarth and Eileen Skelly Frame
9.1 Origin of X-Ray Spectra........................................................................................................................................ 489
9.1.1 Energy Levels in Atoms........................................................................................................................... 489
9.1.2 Moseley’s Law......................................................................................................................................... 494
9.1.3 X-Ray Methods........................................................................................................................................ 494
9.1.3.1 X-Ray Absorption Process...................................................................................................... 495
9.1.3.2 X-Ray Fluorescence Process................................................................................................... 496
9.1.3.3 X-Ray Diffraction Process...................................................................................................... 496
9.2 X-Ray Fluorescence............................................................................................................................................... 498
9.2.1 X-Ray Source........................................................................................................................................... 499
9.2.1.1 X-Ray Tube............................................................................................................................. 499
9.2.1.2 Secondary XRF Sources......................................................................................................... 501
9.2.1.3 Radioisotope Sources.............................................................................................................. 501
9.2.2 Instrumentation for Energy-Dispersive X-Ray Spectrometry................................................................. 504
9.2.2.1 Excitation Source.................................................................................................................... 504
9.2.2.2 Primary Beam Modifiers......................................................................................................... 505
9.2.2.3 Sample Holders....................................................................................................................... 508
9.2.2.4 EDXRF Detectors................................................................................................................... 511
9.2.2.5 Multichannel Pulse Height Analyzer...................................................................................... 514
9.2.2.6 Detector Artifact Escape Peaks and Sum Peaks..................................................................... 515
9.2.3 Instrumentation for Wavelength-Dispersive X-Ray Spectrometry.......................................................... 516
9.2.3.1 Collimators.............................................................................................................................. 516
9.2.3.2 Analyzing Crystals.................................................................................................................. 516
9.2.3.3 Detectors................................................................................................................................. 521
9.2.3.4 Electronic Pulse Processing Units.......................................................................................... 525
9.2.3.5 Sample Changers.................................................................................................................... 527
9.2.4 Simultaneous WDXRF Spectrometers.................................................................................................... 527
9.2.5 Micro-XRF Instrumentation.................................................................................................................... 528
9.2.5.1 Micro-X-Ray Beam Optics..................................................................................................... 528
9.2.5.2 Micro-XRF System Components............................................................................................ 529
9.2.6 Total Reflection XRF............................................................................................................................... 530
9.2.7 Comparison between EDXRF and WDXRF........................................................................................... 530
9.2.8 XRF Applications.................................................................................................................................... 531
9.2.8.1 Analyzed Layer....................................................................................................................... 531
9.2.8.2 Sample Preparation Considerations for XRF.......................................................................... 533
9.2.8.3 Qualitative Analysis by XRF.................................................................................................. 534
xviContents
9.2.8.4 Quantitative Analysis by XRF................................................................................................ 539

9.2.8.5 Applications of Quantitative XRF.......................................................................................... 541
9.3 X-Ray Absorption.................................................................................................................................................. 542
9.3.1 EXAFS..................................................................................................................................................... 546
9.4 X-Ray Diffraction.................................................................................................................................................. 546
9.4.1 Single-Crystal X-Ray Diffractometry...................................................................................................... 548
9.4.2 Crystal Growing....................................................................................................................................... 549
9.4.3 Crystal Structure Determination.............................................................................................................. 549
9.4.4 Powder X-Ray Diffractometry................................................................................................................. 551
9.4.5 Hybrid XRD/XRF Systems..................................................................................................................... 553
9.4.5.1 Compact and Portable Hybrid Systems.................................................................................. 553
9.4.6 Applications of XRD............................................................................................................................... 555
9.4.6.1 Analytical Limitations of XRD............................................................................................... 556
9.5 X-Ray Emission..................................................................................................................................................... 557
9.5.1 Electron Probe Microanalysis.................................................................................................................. 557
9.5.2 Particle-Induced X-Ray Emission........................................................................................................... 558
9.6 Commercial X-Ray Instrument Manufacturers..................................................................................................... 558
Suggested Experiments/Tutorials.......................................................................................................................................559
Problems............................................................................................................................................................................. 561
9.A Appendix: Characteristic X-Ray Wavelengths (Å) and Energies (keV)...................................................................... 566
9.B Appendix: Absorption Edge Wavelengths and Energies.............................................................................................. 571
Bibliography....................................................................................................................................................................... 573
Chapter 10
Mass Spectrometry I: Principles and Instrumentation�� 575
10.1 Principles of MS.................................................................................................................................................... 575
10.1.1 Resolving Power and Resolution of a Mass Spectrometer...................................................................... 579
10.2.1 Sample Input Systems.............................................................................................................................. 580
10.2.1.1 Gas Expansion........................................................................................................................ 580
Inset: A Brief Digression on Units of Measure –Vacuum Systems........................................................ 580
10.2.1.2 Direct Insertion and Direct Exposure Probes.......................................................................... 581
10.2.1.3 Chromatography and Electrophoresis Systems...................................................................... 581
10.2.1.4 Chromatography and Infrared Spectrometry Systems Coupled with
Mass Spectrometry.................................................................................................................. 582
10.2.2 Ionization Sources................................................................................................................................... 582
10.2.2.1 Electron Ionization.................................................................................................................. 582
10.2.2.2 Chemical Ionization................................................................................................................ 583
10.2.2.3 Atmospheric Pressure Ionization Sources.............................................................................. 584
10.2.2.4 Desorption Ionization.............................................................................................................. 586
10.2.2.5 Ionization Sources for Inorganic MS...................................................................................... 592
10.2.3 Mass Analyzers........................................................................................................................................ 593
10.2.3.1 Magnetic and Electric Sector Instruments.............................................................................. 593
10.2.3.2 Time-of-Flight Analyzer......................................................................................................... 596
10.2.3.3 Quadrupole Mass Analyzer..................................................................................................... 601
10.2.3.4 MS/MS and MSn Instruments................................................................................................. 603
10.2.3.5 Quadrupole Ion Trap............................................................................................................... 605
10.2.3.6 Fourier Transform Ion-Cyclotron Resonance......................................................................... 606
10.2.3.7 Orbitrap MS............................................................................................................................ 607
10.2.4 Detectors.................................................................................................................................................. 608
10.2.4.1 Electron Multiplier.................................................................................................................. 609
10.2.4.2 Faraday Cup............................................................................................................................ 610
10.2.4.3 Array Detectors....................................................................................................................... 611
Contents xvii
10.3 Ion Mobility Spectrometry.................................................................................................................................... 611

10.3.1 Handheld DMS JUNO® Chemical Trace Vapor Point Detector.............................................................. 613
10.3.2 Excellims HPIMS-LC System................................................................................................................. 613
10.3.3 Photonis Ion Mobility Spectrometer Engine........................................................................................... 613
10.3.4 SYNAPT G2-S Multistage MS System Incorporating the TriWAVE Ion Mobility Stage...................... 613
10.4 Leaks in Mass Spectrometer Systems.................................................................................................................... 615
Problems............................................................................................................................................................................. 616
Bibliography....................................................................................................................................................................... 617
Chapter 11
Mass Spectrometry II: Spectral Interpretation and Applications�� 619
11.1 Interpretation of Mass Spectra: Structural Determination of Simple Molecules.................................................. 619
11.1.1 Molecular Ion and Fragmentation Patterns.............................................................................................. 621
11.1.2 Nitrogen Rule........................................................................................................................................... 623
11.1.3 Molecular Formulas and Isotopic Abundances........................................................................................ 623
11.1.3.1 Counting Carbon Atoms......................................................................................................... 624
11.1.3.2 Counting Carbon, Nitrogen, and Sulfur Atoms...................................................................... 625
11.1.3.3 Counting Oxygen Atoms........................................................................................................625
11.1.4 Compounds with Heteroatoms................................................................................................................ 626
11.1.5 Halogen Isotopic Clusters........................................................................................................................ 627
11.1.6 Rings Plus Double Bonds........................................................................................................................ 630
11.1.7 Common Mass Losses on Fragmentation................................................................................................ 631
11.2 Mass Spectral Interpretation: Some Examples...................................................................................................... 631
11.2.1 Mass Spectra of Hydrocarbons................................................................................................................ 635
11.2.2 Mass Spectra of Other Organic Compound Classes................................................................................ 640
11.2.2.1 Alcohols.................................................................................................................................. 640
11.2.2.2 Ethers...................................................................................................................................... 642
11.2.2.3 Ketones and Aldehydes........................................................................................................... 642
11.2.2.4 Carboxylic Acids and Esters................................................................................................... 645
11.2.3 Compounds Containing Heteroatoms...................................................................................................... 646
11.2.3.1 Nitrogen-Containing Compounds........................................................................................... 646
11.2.3.2 Sulfur-Containing Compounds............................................................................................... 647
11.3 Applications of Molecular MS.............................................................................................................................. 649
11.3.1 High-Resolution Mass Spectrometry....................................................................................................... 649
11.3.1.1 Achieving Higher Mass Accuracy (but Not Resolution) from Low-Resolution
MS Instruments....................................................................................................................... 650
11.3.1.2 Improving the Quantitation Accuracy of Isotope Ratios from Low-Resolution
MS Instrument Data Files....................................................................................................... 651
11.3.2 Quantitative Analysis of Compounds and Mixtures................................................................................ 652
11.3.3 Protein-Sequencing Analysis (Proteomics)............................................................................................. 654
11.3.4 Gas Analysis............................................................................................................................................ 655
11.3.5 Environmental Applications....................................................................................................................655
11.3.6 Other Applications of Molecular MS...................................................................................................... 657
11.3.7 Limitations of Molecular MS.................................................................................................................. 657
11.4 Atomic MS............................................................................................................................................................. 657
11.4.1 ICP-MS.................................................................................................................................................... 657
11.4.2 Applications of Atomic MS..................................................................................................................... 660
11.4.2.1 Geological and Materials Characterization Applications....................................................... 661
11.4.2.2 Speciation by Coupled Chromatography-ICP-MS................................................................. 663
11.4.2.3 Applications in Food Chemistry, Environmental Chemistry, Biochemistry,
Clinical Chemistry, and Medicine........................................................................................... 664
11.4.2.4 Coupled Elemental Analysis MS............................................................................................ 666
11.4.3 Interferences in Atomic MS..................................................................................................................... 666
11.4.3.1 Matrix Effects......................................................................................................................... 666
xviiiContents
11.4.3.2 Spectral (Isobaric) Interferences............................................................................................. 667

11.4.4 Instrumental Approaches to Eliminating Interferences........................................................................... 670
11.4.4.1 High-Resolution ICP-MS (HR-ICP-MS)................................................................................ 670
11.4.4.2 Collision and Reaction Cells................................................................................................... 670
11.4.4.3 MS/MS Interference Removal................................................................................................ 671
11.4.5 Limitations of Atomic MS....................................................................................................................... 672
11.5 Common Spurious Effects in Mass Spectrometry................................................................................................. 674
Problems............................................................................................................................................................................. 674
11.A Appendix.................................................................................................................................................................... 682
Bibliography....................................................................................................................................................................... 684
Chapter 12
Principles of Chromatography�� 687
12.1 Introduction to Chromatography........................................................................................................................... 687
12.2 The Nature of Mixtures......................................................................................................................................... 688
12.3 What Is the Chromatographic Process?................................................................................................................. 691
12.4 Chromatography in More than One Dimension.................................................................................................... 692
12.5 Visualization of the Chromatographic Process at the Molecular Level: Analogy to “People on a
Moving Belt Slideway”.......................................................................................................................................... 693
12.6 Digression on the Central Role of Silicon–Oxygen Compounds in Chromatography.......................................... 696
12.7 Basic Equations Describing Chromatographic Separations.................................................................................. 698
12.8 How Do Column Variables Affect Efficiency (Plate Height)?............................................................................... 701
12.9 Practical Optimization of Chromatographic Separations...................................................................................... 702
12.10 Extra-Column Band Broadening Effects............................................................................................................... 703
12.11 Qualitative Chromatography: Analyte Identification............................................................................................. 704
12.12 Quantitative Measurements in Chromatography................................................................................................... 704
12.12.1 Peak Area or Peak Height: What Is Best for Quantitation?..................................................................... 705
12.12.2 Calibration with an External Standard..................................................................................................... 705
12.12.3 Calibration with an Internal Standard...................................................................................................... 706
12.13 Examples of Chromatographic Calculations......................................................................................................... 707
Problems............................................................................................................................................................................. 708
Bibliography....................................................................................................................................................................... 709
Chapter 13
Gas Chromatography�� 711
13.1 Historical Development of GC: The First Chromatographic Instrumentation...................................................... 711
13.2 Advances in GC Leading to Present-Day Instrumentation.................................................................................... 712
13.3 GC Instrument Component Design (Injectors)...................................................................................................... 713
13.3.1 Syringes................................................................................................................................................... 713
13.3.2 Autosamplers........................................................................................................................................... 714
13.3.3 SPME....................................................................................................................................................... 714
13.3.4 Split Injections......................................................................................................................................... 715
13.3.5 Splitless Injections................................................................................................................................... 715
13.4 GC Instrument Component Design (the Column)................................................................................................. 716
13.4.1 Column Stationary Phase......................................................................................................................... 716
13.4.2 Selecting a Stationary Phase for an Application...................................................................................... 719
13.4.3 Effects of Mobile Phase Choice and Flow Parameters............................................................................ 721
13.5 GC Instrument Operation (Column Dimensions and Elution Values)................................................................... 722
13.6 GC Instrument Operation (Column Temperature and Elution Values).................................................................. 724
13.7 GC Instrument Component Design (Detectors).................................................................................................... 728
13.7.1 Thermal Conductivity Detector............................................................................................................... 730
13.7.2 Flame Ionization Detector....................................................................................................................... 730
13.7.3 Electron Capture Detector....................................................................................................................... 732
13.7.4 Electrolytic Conductivity Detector.......................................................................................................... 734
Contents xix
13.7.5 Sulfur–Phosphorus Flame Photometric Detector.................................................................................... 734

13.7.6 Sulfur Chemiluminescence Detector....................................................................................................... 734
13.7.7 Nitrogen–Phosphorus Detector................................................................................................................ 735
13.7.8 Photoionization Detector......................................................................................................................... 735
13.7.9 Helium Ionization Detector..................................................................................................................... 736
13.7.10 Atomic Emission Detector....................................................................................................................... 737
13.8 Hyphenated GC Techniques (GC-MS, GC-IR, GC-GC, or 2D GC)..................................................................... 737
13.8.1 GC-MS..................................................................................................................................................... 737
13.8.2 GC-IR Spectrometry................................................................................................................................ 740
13.8.3 Comprehensive 2D Gas Chromatography (GC × GC or GC2)................................................................ 740
13.9 Retention Indices (A Generalization of Relative Rt Information).........................................................................743
13.10 Scope of GC Analyses........................................................................................................................................... 744
13.10.1 GC Behavior of Organic Compound Classes.......................................................................................... 744
13.10.2 Derivatization of Difficult Analytes to Improve GC Elution Behavior................................................... 744
13.10.3 Gas Analysis by GC................................................................................................................................. 745
13.10.4 Limitations of GC.................................................................................................................................... 746
Problems............................................................................................................................................................................. 746
13.A Appendix: GC Internet Web Resources..................................................................................................................... 748
Bibliography....................................................................................................................................................................... 749
Chapter 14
Chromatography with Liquid (and Fluid) Mobile Phases�� 751
14.1 High-Performance Liquid Chromatography.......................................................................................................... 751
14.1.1 HPLC Column and Stationary Phases..................................................................................................... 751
14.1.1.1 Support Particle Considerations.............................................................................................. 753
14.1.1.2 Stationary-Phase Considerations............................................................................................ 755
14.1.1.3 Chiral Phases for Separation of Enantiomers......................................................................... 755
14.1.2 Effects on Separation of Composition of the Mobile Phase.................................................................... 756
14.1.2.1 New HPLC-Phase Combinations for Assays of Very Polar Biomolecules............................. 757
14.1.2.2 Ion-Exchange Chromatography.............................................................................................. 758
14.1.2.3 Hydrophilic Interaction Liquid Chromatography................................................................... 758
14.1.2.4 Polar-Embedded Long Organic Chains on Silica................................................................... 758
14.1.2.5 Polar-Endcapped Short Hydrocarbon Chains on Silica.................................................... 758
14.1.3 Design and Operation of an HPLC Instrument........................................................................................ 758
14.1.4 HPLC Detector Design and Operation.................................................................................................... 761
14.1.4.1 Refractive Index Detector....................................................................................................... 762
14.1.4.2 Evaporative Light-Scattering Detector................................................................................... 763
14.1.4.3 UV/VIS Absorption Detectors................................................................................................ 765
14.1.4.4 Fluorescence Detectors........................................................................................................... 767
14.1.4.5 Electrochemical Detectors...................................................................................................... 768
14.1.4.6 Conductometric Detectors...................................................................................................... 770
14.1.4.7 Charge Detector (QD) [Thermo Scientific]............................................................................ 770
14.1.4.8 Summary Comparison of Six Major HPLC Detectors........................................................... 771
14.1.5 Derivatization in HPLC........................................................................................................................... 771
14.1.6 Hyphenated Techniques in HPLC............................................................................................................ 773
14.1.6.1 Interfacing HPLC to Mass Spectrometry................................................................................ 773
14.1.7 Applications of HPLC............................................................................................................................. 777
14.1.7.1 Biochemical Applications in Proteomics................................................................................ 778
14.2 Chromatography of Ions Dissolved in Liquids...................................................................................................... 782
14.2.1 Ion Chromatography................................................................................................................................ 784
14.2.1.1 Single-Column IC................................................................................................................... 787
14.2.1.2 Indirect Detection in IC.......................................................................................................... 787
14.3 Affinity Chromatography....................................................................................................................................... 788
14.4 Size-Exclusion Chromatography........................................................................................................................... 789
14.5 Supercritical Fluid Chromatography..................................................................................................................... 791
xxContents
14.5.1 Operating Conditions............................................................................................................................... 792

14.5.2 Effect of Pressure..................................................................................................................................... 792
14.5.3 Stationary Phases..................................................................................................................................... 792
14.5.4 Mobile Phases.......................................................................................................................................... 792
14.5.5 Detectors.................................................................................................................................................. 792
14.5.6 SFC versus Other Column Methods........................................................................................................ 792
14.5.7 Applications............................................................................................................................................. 793
14.5.8 Ultra Performance Convergence Chromatography: A New Synthesis.................................................... 793
14.6 Electrophoresis...................................................................................................................................................... 794
14.6.1 Capillary Zone Electrophoresis (CZE).................................................................................................... 794
14.6.2 Sample Injection in CZE......................................................................................................................... 798
14.6.3 Detection in CZE..................................................................................................................................... 799
14.6.4 Modes of CE............................................................................................................................................ 800
14.6.4.1 CZE......................................................................................................................................... 800
14.6.4.2 Capillary Gel Electrophoresis................................................................................................. 801
14.6.4.3 Capillary Isoelectric Focusing................................................................................................ 801
14.6.5 Capillary Electrochromatography............................................................................................................ 803
14.6.5.1 Micellar Electrokinetic Capillary Chromatography................................................................ 803
14.7 Planar Chromatography and Planar Electrophoresis............................................................................................. 805
14.7.1 Thin-Layer Chromatography................................................................................................................... 805
14.7.1.1 Parallel 1D TLC Separations.................................................................................................. 805
14.7.1.2 Detection in TLC.................................................................................................................... 806
14.7.1.3 2D Separations Using TLC (2D-TLC)...................................................................................808
14.7.2 Planar Electrophoresis on Slab Gels........................................................................................................ 808
14.7.2.1 1D Planar Gel Electrophoresis................................................................................................ 808
14.7.2.2 2D Planar Gel Electrophoresis................................................................................................ 808
Problems and Exercises...................................................................................................................................................... 810
14.A Appendix LC/CE/TLC Internet Web Resources........................................................................................................ 811
Bibliography....................................................................................................................................................................... 812
Chapter 15
Electroanalytical Chemistry�� 815
15.1 Fundamentals of Electrochemistry........................................................................................................................ 815
15.2 Electrochemical Cells............................................................................................................................................ 816
15.2.1 Line Notation for Cells and Half-Cells.................................................................................................... 819
15.2.2 Standard Reduction Potentials................................................................................................................. 819
15.2.2.1 Standard Hydrogen Electrode................................................................................................. 819
15.2.3 Sign Conventions..................................................................................................................................... 821
15.2.4 Nernst Equation....................................................................................................................................... 821
15.2.5 Activity Series.......................................................................................................................................... 822
15.2.6 Reference Electrodes............................................................................................................................... 823
15.2.6.1 Saturated Calomel Electrode................................................................................................... 823
15.2.6.2 Silver/Silver Chloride Electrode............................................................................................. 824
15.2.7 Electrical Double Layer........................................................................................................................... 824
15.3 Electroanalytical Methods..................................................................................................................................... 825
15.3.1 Potentiometry........................................................................................................................................... 826
15.3.1.1 Indicator Electrodes................................................................................................................ 827
15.3.1.2 Instrumentation for Measuring Potential................................................................................ 833
15.3.1.3 Analytical Applications of Potentiometry............................................................................... 835
15.3.2 Coulometry.............................................................................................................................................. 843
15.3.2.1 Electrogravimetry...................................................................................................................845
15.3.2.2 Instrumentation for Electrogravimetry and Coulometry......................................................... 845
15.3.2.3 Applied Potential.................................................................................................................... 845
15.3.2.4 Analytical Determinations Using Faraday’s Law................................................................... 846
15.3.3 Conductometric Analysis......................................................................................................................... 850
Contents xxi
15.3.3.1 Instrumentation for Conductivity Measurements................................................................... 852

15.3.3.2 Analytical Applications of Conductometric Measurements................................................... 853
15.3.4 Polarography............................................................................................................................................ 855
15.3.4.1 Classical or DC Polarography................................................................................................. 856
15.3.4.2 Half-Wave Potential................................................................................................................ 859
15.3.4.3 Normal Pulse Polarography.................................................................................................... 860
15.3.4.4 Differential Pulse Polarography.............................................................................................. 861
15.3.5 Voltammetry............................................................................................................................................863
15.3.5.1 Instrumentation for Voltammetry............................................................................................ 865
15.3.5.2 Stripping Voltammetry............................................................................................................ 865
15.3.5.3 pHit Sensor for pH by Cyclic Voltammetry on Carbon.......................................................... 866
15.3.5.4 Applications of Anodic Stripping Voltammetry...................................................................... 867
15.4 Liquid Chromatography Detectors........................................................................................................................ 868
15.4.1 Voltammetric Detection........................................................................................................................... 868
15.4.2 Conductometric Detection....................................................................................................................... 869
15.5 Spectroelectrochemistry........................................................................................................................................ 870
15.6 Quartz Crystal Microbalance................................................................................................................................. 872
15.6.1 Piezoelectric Effect.................................................................................................................................. 872
15.6.2 Electrochemical Quartz Crystal Microbalance........................................................................................ 876
15.7 Electrochemistry at the Nanoscale......................................................................................................................... 876
Problems............................................................................................................................................................................. 879
15.A Appendix: Selected Standard Reduction Potentials at 25 °C..................................................................................... 880
Bibliography....................................................................................................................................................................... 880
Chapter 16
Surface Analysis�� 883
16.1 Introduction............................................................................................................................................................ 883
16.2 Electron Spectroscopy Techniques........................................................................................................................ 883
16.2.1 X-Ray Photoelectron Spectroscopy......................................................................................................... 885
16.2.1.1 Instrumentation for XPS......................................................................................................... 886
16.2.1.2 Sample Introduction and Handling for Surface Analysis....................................................... 890
16.2.1.3 Analytical Applications of XPS.............................................................................................. 891
16.2.2 Auger Electron Spectroscopy.................................................................................................................. 898
16.2.2.1 Instrumentation for AES......................................................................................................... 899
16.2.2.2 Applications of AES............................................................................................................... 900
16.3 Ion Scattering Spectroscopy.................................................................................................................................. 902
16.4 Secondary Ion Mass Spectrometry........................................................................................................................ 905
16.4.1 Instrumentation for SIMS........................................................................................................................ 906
16.4.1.1 Primary Ion Sources................................................................................................................ 906
16.4.1.2 Mass Spectrometers................................................................................................................ 907
16.4.2 Analytical Applications of SIMS............................................................................................................. 907
16.4.2.2 Ion Microprobe Mass Spectrometry....................................................................................... 909
16.5 Electron Microprobe (Electron Probe Microanalysis)........................................................................................... 910
Problems............................................................................................................................................................................. 910
Bibliography....................................................................................................................................................................... 911
Chapter 17
Thermal Analysis�� 913
17.1 Thermogravimetry................................................................................................................................................. 914
17.1.1 TGA Instrumentation............................................................................................................................... 916
17.1.2 Analytical Applications of Thermogravimetry........................................................................................ 920
17.1.3 Derivative Thermogravimetry.................................................................................................................. 924
17.1.4 Sources of Error in Thermogravimetry.................................................................................................... 926
xxiiContents
17.2 Differential Thermal Analysis...............................................................................................................................926

17.2.1 DTA Instrumentation............................................................................................................................... 927
17.2.2 Analytical Applications of DTA.............................................................................................................. 929
17.3 Differential Scanning Calorimetry......................................................................................................................... 930
17.3.1 DSC Instrumentation............................................................................................................................... 930
17.3.2 Applications of DSC................................................................................................................................ 934
17.3.2.1 Pressure DSC.......................................................................................................................... 936
17.3.2.2 Modulated DSC...................................................................................................................... 937
17.4 Hyphenated Techniques......................................................................................................................................... 938
17.4.1 Hyphenated Thermal Methods................................................................................................................938
17.4.2 Evolved Gas Analysis.............................................................................................................................. 938
17.5 Thermometric Titrimetry....................................................................................................................................... 941
17.5.1 Applications of Thermometric Titrimetry...............................................................................................944
17.6 Direct Injection Enthalpimetry.............................................................................................................................. 944
17.7 Microcalorimetry................................................................................................................................................... 945
17.7.1 Micro-DSC Instrumentation.................................................................................................................... 945
17.7.2 Applications of Micro-DSC..................................................................................................................... 946
17.7.3 Isothermal Titration Calorimetry............................................................................................................. 946
17.7.4 Microliter Flow Calorimetry.................................................................................................................... 948
17.8 Thermomechanical Analysis and Dynamic Mechanical Analysis......................................................................... 949
17.8.1.1 TMA Equipment..................................................................................................................... 952
17.8.1.2 DMA Equipment..................................................................................................................... 953
17.8.2 Applications of TMA and DMA.............................................................................................................. 954
17.9 Optical Thermal Analysis...................................................................................................................................... 957
17.9.1 Heating Microscope................................................................................................................................. 957
17.9.2 Optical Dilatometers................................................................................................................................ 958
17.9.3 Optical Thermal Analyzers with Differential Thermal Analysis............................................................. 960
17.10 Summary................................................................................................................................................................ 962
Problems............................................................................................................................................................................. 964
Bibliography....................................................................................................................................................................... 967
Appendix: Abbreviations, Acronyms, and Symbols�� 969

Subject Index�� 977
Chemical Index�� 987
Preface to the Eighth Edition
Analytical chemistry today is almost entirely instru- and MS). The NMR, IR, and MS spectra, courtesy of Bio-
mental analytical chemistry, performed by many scientists, Rad Laboratories, Informatics Division (IR, NMR), Aldrich
engineers, and technicians, many of whom are not chemists. Chemical Company (NMR), Agilent Technologies, Inc., and
Analytical instrumentation is crucial to research in molecular the NIST Chemistry Webbook were obtained on modern
biology, medicine, geology, food science, materials science, instruments to reflect what students will encounter in modern
forensics, and a myriad of other fields. While it is true that it laboratories. Additional NMR spectra have been provided
is no longer necessary to have almost artistic skills to obtain by Bruker Corporation and picoSpin LLC (now Thermo
accurate and precise analytical results using instrumentation, Fisher Scientific). The use of spreadsheets for performing
the instruments cannot be considered “black boxes” by those calculations has been introduced with examples. Reflecting
using them. “Garbage in, garbage out” holds true for analytical the ubiquitous nature of the Internet, we have included a
instrumentation as well as computers. The intent of this book large number of instrument manufacturers’ websites and
is to provide users with an understanding of their instruments. government websites, which contain extensive resources
In keeping with the earlier editions of this text, the book for interested students. This includes an expanded discus-
is designed for teaching undergraduates and those with no sion of standard reference materials (available from National
instrumental analytical chemistry background how modern Measurement Laboratories) and standard reference data.
analytical instrumentation works and what the uses and Sampling, sample handling, and storage and sample
limitations of analytical instrumentation are. Mathematics preparation methods are extensively covered, and modern
beyond algebra is kept to a minimum. Prior editions touted methods such as accelerated solvent extraction, solid-phase
the need for no background in calculus, physics, or physical microextraction (SPME), QuEChERS, and microwave
chemistry, but we recognize that some familiarity in these techniques are included. Instrumentation, the analysis of
areas is helpful. Indeed, it is unrealistic to assume that a liquids and solids, and applications of NMR are discussed
student taking analytical instrumentation will have no such in detail. A section on hyphenated NMR techniques is
background and doing so is a disservice to them. Here, we included, along with an expanded section on MRI and
do not sell them short; we give them the opportunity to build advanced imaging. The IR instrumentation section is
upon their background. focused on FTIR instrumentation, and a discussion of
The major fields of modern instrumentation are covered, photoacoustic spectroscopy is now included. Absorption,
including applications of each type of instrumental tech- emission, and reflectance spectroscopy are discussed, as is
nique. Each chapter includes a discussion of the fundamental FTIR microscopy. ATR has been expanded. Near-IR instru-
principles underlying each technique, detailed descriptions mentation and applications are presented, and the topic of
of the instrumentation, and a large number of applications. chemometrics is introduced. Coverage of Raman spectros-
Each chapter includes an updated bibliography and practice copy includes resonance Raman, surface-enhanced Raman,
problems, and most chapters have suggested experiments and Raman microscopy. Chemical imaging is described,
appropriate to the technique. including confocal Raman imaging. UV and visible spec-
This edition has been completely updated, revised, and troscopy includes innovations such as flow-through sample
expanded. To match the preference of most instructors, the holders and fiber-optic probes, as well as instruments for
order of presentation has been changed from that of the sev- analysis of submicroliter volumes and nondestructive ana-
enth edition. Now, after the introduction to spectroscopy, lysis for nucleic acid and protein determinations. UV absorp-
UV-Vis is discussed, and naturally, instructors are free to tion spectral interpretation for organic molecules is covered
change the order of presentation once the fundamentals have in depth. Applications described include nucleic acid and
been introduced. The topics of chromatography and mass protein measurements, spectrophotometric titrations, and
spectrometry have been greatly expanded to better reflect new applications in forensic chemistry. Nephelometry, tur-
the predominance of chromatography and mass spectrom- bidimetry, fluorescence, and phosphorescence are described
etry instrumentation in modern laboratories. The equally in detail, including instrumentation and applications. The
important topic of NMR, expanded in the last edition to measurement of color using the CIE system is described
focus on FTNMR, 13C, and 2D NMR spectral interpretation, with examples.
now includes time domain NMR (relaxometry) and an over- Mass spectrometry is treated as two chapters and
view of low-field, benchtop, and miniature instrumentation. covers both organic and inorganic MS instrumentation and
The topic of electron spin resonance spectroscopy (ESR, applications. We dispel the often confusing mix of units
EPR) has been added due to the recent availability of small, that describe vacuum systems, a topic that is applicable to
low-cost ESR instrumentation and its impact on materials other chapters as well. GC-MS and LC-MS are described
characterization and bioanalysis. along with MSn instruments for organic and inorganic MS,
A unique feature of this text is the combination of instru- including new triple quad ICP-MS instrumentation. Modern
mental analysis with organic spectral interpretation (IR, NMR, ionization methods such as electrospray and MALDI are
xxiii
xxiv Preface to the Eighth Edition
also described. Surface scanning and ionization sources, porous, core–shell–particle stationary phases, which enable
such as desorption electrospray ionization (DESI) and laser much higher resolution and faster analysis. Such UHPLC
ablation electrospray ionization (LAESI), and the DART methods now constitute more than half of the LC methods
atmospheric pressure ionization source are introduced. in operation. Detailed discussions of instrumental design and
More advanced high- resolution systems, such as the the operation of new detectors, such as the charge detector for
Orbitrap, and long-path time of flight (JEOL SpiralTOF and ion chromatography, the corona discharge detector, pulsed
LECO Citius-LC and Pegasus GC-HRT) are described. The amperometric detector for saccharides, and the evaporative
use of MS peak profile correction software to improve mass light-scattering detector for spectrally inert analytes have
and spectral accuracy from lower resolution MS instru- been added. Major sections on the design and operation of
mentation is described, and important cautions in the use HPLC interfaces to mass spectrometers (ESI and APCI) have
of search software are discussed. Uses of HRMS receive been added, and examples of their use in protein or peptide
increased attention. A new section on ion mobility spec- sequencing and identification are included. Tables of amino
trometry is added, with a discussion of four systems ranging acid structures and nomenclature have been included so that
from handheld vapor monitors to stages enabling charac- students can follow these descriptions. Separate sections on
terization of different conformational isomers in top-end ion chromatography, affinity chromatography, size exclusion
proteomics analyzers. Organic mass spectral interpretation chromatography, and supercritical fluid extraction and chro-
is covered with many examples and new spectra. Organic matography (SFE/C) have been expanded. The revival of
and inorganic applications focus on speciation using GC- analytical SFC by the very recent redesign of instrumentation
MS, LC-MS, and hyphenated ICP-MS, with an emphasis to enable ultra- performance convergence chromatography
on proteomics, biomolecules, and species of environ- (UPC2) is introduced, and examples of the improvements
mental interest. Examples of atomic MS applications using it offers are given. Planar and capillary electrophoresis are
new simultaneous and “triple quad” ICP-MS systems and described in detail in Chapter 13, despite not being strictly
glow discharge surface sampling and hydrogen in metals defined as chromatographic methods. Examples are given
profiling are added. All major modern atomic absorption of the use of capillary electrophoresis with fluorescence-
and emission techniques and instrumentation are covered, derivatization detection to gene sequencing in genomics
including new MP- AES and triple quadrupole ICP- MS and of 2D slab-gel, isoelectric focusing/SDS-PAGE elec-
instruments. trophoresis for protein peptide mapping in proteomics. New
Chromatographic separations and instrumentation are appendices provide links to websites containing examples of
treated in three chapters which begin with a fundamental thousands of chromatographic separations and encourage the
description of mixtures from a molecular view. The first of student to learn how to utilize the resources of commercial
these chapters covers the nature of the chromatographic pro- column vendors to find a solution to particular separation or
cess, starting with the nature of mixtures. A minimum of measurement problems.
complex formulas is introduced; instead, extensive descrip- The discussion of XPS has been revised to reflect
tion and analogy are employed to give the student an intuitive the current state of commercial instrumentation.
grasp of the mechanisms giving rise to separation, reso- Spectroelectrochemistry and nanoelectrochemistry topics
lution, and detection of separated components. Additional have been expanded, with commercial instrumentation and
graphics have been added to clarify the concepts. In line with examples. Probes based on quartz crystal microbalance
current practice, GC is treated as a method almost completely response are covered. The radical new pH meter design
employing open tubular (capillary) columns. We treat SPME using calibration- free, easily stored, extremely robust,
injection, low-bleed stationary phases, ionic liquid stationary voltammetric-based probes with surfaces of anthraquinone-
phases, compound-selective detectors, and especially inter- bonded multiwalled carbon nanotubes is described. The
facing to spectrometric detectors and the use of computerized important bioanalytical technique of microcalorimetry has
data processing. So-called hyphenated techniques such as been added to Chapter 17, Thermal Analysis, and TMA and
GC-MS, GC-IR, GC-IR-MS, and comprehensive GC-GC are DMA have been greatly expanded, thanks to the excellent
included, and new sections on retention indices, derivatization materials and invaluable assistance from TA Instruments.
to improve detectability and volatility, and analysis of gases Optical thermal instrumentation is now covered.
in air or water have been added. The requirements for We are pleased that in this edition we have included
implementing and instrumentalizing HPLC are developed both a subject index and a chemical index. While the chem-
from the preceding discussion of GC. The student gains an ical index is not exhaustive, it will allow the reader to rap-
appreciation of the difficulties that caused this instrumentation idly find analytical metrology for many compounds and
to lag behind GC. Once overcome, liquid chromatographic elements.
instrumentation is seen to have wider applicability, especially While the authors are extremely grateful to the many
in the burgeoning subdisciplines of bioanalysis, proteomics, experts listed in the acknowledgments, who have provided
and genomics. Coverage includes the use of superficially graphics, technical advice, rewrites, and reviews of various
Preface to the Eighth Edition xxv
sections, any errors that are present are entirely the respon- expanded discussion of uncertainty in the eighth edition,
sibility of the authors. sensitivity factors are expressed with derivatives. Don’t be
intimidated: simple calculus is a lot like cooking —you
Thomas J. Bruno simply follow a recipe.
James W. Robinson Unique among all instrumental analysis texts we expli-
Eileen M. Skelly Frame citly discuss safety, up front in Chapter 2. The presenta-
George M. Frame II tion intentionally avoids a finger-wagging, thou-shalt-not
approach in favor of a how-to discussion of good laboratory
and industrial practice. It is focused on hazards (and rem-
TO THE STUDENT OR READER: edies) that might be encountered in the use of instrumenta-
tion; we don’t even mention chemical safety (which should
This book is unique among texts on analytical instru- have been emphasized for you before now). The informa-
mentation in that the discussion of each technique starts tion in Chapter 2 will not only help you complete the course
with the underlying atomic or molecular picture and then safely, it will also be valuable material for you as a life skill.
develops the level of sophistication that is needed to make This chapter might not be discussed in class, but please read
each method work for you. Yes, you have heard this before, it anyway.
but this book actually delivers. What does this mean? Each
student approaches the study of analytical instrumentation
with some knowledge from prior course work, but the com- TO THE INSTRUCTOR:
pleteness of that preparation will depend on the interests
of your instructors. If, for example, your organic instructor We realize you are busy so we will make this short
was not interested in ultraviolet spectroscopy, they likely and sweet.
spent little time on it. Here, on the other hand, you will start Past editions of this book touted the need for minimal
with the molecular infrastructure, and the development and mathematics knowledge and skills. In the greatly expanded
application of the method will therefore make sense to you. discussion of uncertainty presented in the eighth edition,
This is a book that you will want to keep in your pro- sensitivity factors are expressed as derivatives (typically
fessional toolbox rather than selling back to the bookstore from introductory calculus). Understand that it is still pos-
when the course is done. We appreciate that your textbooks sible to approach error and uncertainty with a formulae
are costly, but because of our approach in this book, you will approach, and sidestep expanded uncertainty, but here you
be able to go back to it in a few years, pick it up, and in short can use either approach.
order be up to speed. If you go on to an advanced degree, The chapter on safety is meant to augment your
this aspect will be indispensable to you. But, even if you go institution’s safety program and standard operating
into medicine or law or some other profession, the case may procedures. If you do not discuss this chapter in lecture,
arise in which an NMR spectrum or a mass spec will have we ask that you assign it as required reading. Or simply
bearing, and you will want to revisit the topic. This book highlight the points relevant to your program. If you are a
will get you there quickly because we start simple and don’t new teaching assistant doing this for the first time, you will
assume anything. The added chemical index will allow you have to know this material in your sleep; it is simply part
to rapidly find analytical metrology and procedures for of the job.
many compounds and elements. You will note that the order of presentation has been
Prior editions of this book touted minimization of math- changed in the eighth edition such that UV-Vis and vibra-
ematics and absence of calculus, and yes, you can use this tional spectrometry follows introductory spectroscopy, then
book with only a bit of rudimentary algebra. But honestly, NMR and AAS and AES. We think this is a more natural
it is not really possible to appreciate uncertainty analysis progression, but feel free to modify the presentation to suit
without taking a derivative now and then, so in the greatly your students’ needs.
Acknowledgments
The following people are gratefully acknowledged for Professor Gary Siudzak, Scripps Research Institute
their assistance in the successful completion of this edition. Center for Mass Spectrometry; Dr. Robert Kobelski,
They provided diagrams, photographs, application notes, Centers for Disease Control, Atlanta, Georgia; Professor
spectra, chromatograms, and many helpful comments and David Hercules, Vanderbilt University; Dr. S.E. Stein and
suggestions regarding the text. Thanks are due, in no spe- Anzor I. Mikaia, NIST Mass Spec Data Center; Volker
cific order, to Birke Götz, BÜCHI Labortechnik AG; the Thomsen, NITON Corp.; Dr. Gilles Widawski and Fumi
entire TA Instruments team with special thanks to Roger Akimaru, NETZSCH Instruments North America, LLC;
Blaine, Fred Wiebke, Charles Potter, and Terry Allen; Gwen Dr. Thomas Rampke and Stephan Knappe, NETZSCH-
Boone, Doug Shrader, Ed McCurdy, Pat Grant, Laima Gerätebau GmbH; Michael Fry, Dr. Chip Cody, Pam
Baltusis, Dan Steele, Jim Simon, Paul Canavan, Amy Mansfield, Masaaki Ubukata, and Patricia Corkum, JEOL,
Herlihy, Eric Endicott, Valerie Lopez, William Champion, Inc.; Andy Rodman, Giulia Orsanigo, Christopher Tessier,
and Matt Nikow, Agilent Technologies; Dr. A. Horiba, Sarah Salbu, and Danielle Hawthorne, PerkinElmer,
Atsuro Okada, Juichiro Ukon, Mike Pohl, Philippe Hunault, Inc.; Nancy Fernandes, Newport Corporation; Ralph
Patrick Chapon, Phil Shymanski, Diane Surine, Joanne Obenauf, SPEX CertiPrep, Inc.; Rolf Schlake, Applied
Lowy, Andrew Whiteley, Christophe Morin, and David Separations, Inc.; Paul Smaltz and Anne Logan, Avantor
Tuschel, HORIBA Scientific; Peter Brown, Dave Pfeil, and Performance Materials, Inc.; Donald Bouchard, Anasazi
Dr. Manuel Almeida, Teledyne Leeman Labs, Inc.; Steve Instruments, Inc.; John Hammond, Rosemary Huett, and
Sauerbrunn, Mettler Toledo, Inc.; Mark Mabry, Bob Coel, Keith Hulme, Starna Scientific, Inc.; John Price, Dean
Ed Oliver, Lara Pryde, Jim Ferrara, John Flavell, Chuck Antic, and Chuck Miller, picoSpin, LLC; Jenni L. Briggs
Douthitt, Keith Bisogno, Mary Meegan- Litteer, Jackie and Z. Stanek, PIKE Technologies; Dawn Nguyen,
Lathos-Markham, Todd Strother, Joseph Dorsheimer, Marty Enwave Optronics, Inc.; Alicia Kimsey, Claire Dentinger,
Palkovic, Dr. Julian Phillips, Wendy Weise, Carl Millholland, Rigaku Raman Technologies, Inc.; Steve Pullins and
Michael Bradley, Janine O’Rourke, Eric Francis, Art Eric Bergles, BaySpec, Inc.; Evan Friedmann, Hellma
Fitchett, Dr. Stephan Lowry, Timothy O. Deschaines, USA, Inc.; Eric Shih, Mark Salerno, C Technologies;
Fergus Keenan, Simon Nunn, Ryan Kershner, Elizabeth Monique Claverie, Dr. Joseph E. Johnson, and Dr. David
Guiney, Russell Diemer, Michael W. Allen, Bill Sgammato, Wooton, Microspectral Analysis, LLC; Keith Long, Mark
Dr. John Wolstenholme, Dr. Joachim Hinrichs, Carolyn Talbott, Mark Taylor, and Kevin McLaughlin, Herb Pottoff,
Carter, Kathy Callaghan, Allen Pierce, Arthur Fitchitt, Shimadzu Scientific Instruments, Inc.; Steve Buckley
Fraser McLeod, and Frank Hoefler, Thermo Fisher and Edwin Pickins, TSI, Inc.; Chris Beasley, Burak Uglut,
Scientific; Mary Allen, Allen Design; Todd Maxwell, Gamry Instruments; Mark Handley, International
CETAC Technologies; David Coler, PANalytical, Inc.; Crystal Manufacturing, Inc.; Eoin Vincent, Samuel
William Ickes, Buchi Corporation; Kenneth Krewson, Machado, and John Nikitas, Olympus NDT; Dr. John
Supercritical Fluid Technologies, Inc.; Jim McKinley, C. Edwards, Process NMR Associates, LLC; Mary Jo
Dale Edcke, Bob Anderhalt, AMETEK; Mike Hurt and Luis Wojtusik and Michele Giordiano, GE Healthcare Life
Moreno, Hitachi High Technologies America; Michael Sciences; Bryan C. Webb, Norton Scientific Inc.; Ryan
Nelson, Rigaku Corporation; Robert Bartek, Applied Brennan, Glass Expansion, Inc.; Heather Grael, Implen,
Rigaku Technologies, Inc.; Dr. Tom Dulski, Carpenter Inc.; Ray Wood, AstraNet Systems, Ltd.; Jamie Huffnagle,
Technologies; Dr. Alex Medek, Pfizer Central Research Len Morrisey, and Brent Cleveland, ASTM International;
and Development; Dr. James Roberts, Lehigh University; Brian J. Murphy and Dave DePasquale, Waters Corp.;
Dr. Thorsten Thiel, Kodi Morton, Catherine Fisk, Andrew Joseph H. Kennedy, Prosolia; Carol Morloff, Excellims;
Hess, Armin Gross, Sarah Nelson, and Jerry Sooter, Mark Margaret M. Cooley, Photonis; Stanton Loh, Palisade;
Chaykovsky, Bruker Corporation; Pat Wilkinson and Mike Festa, IonSense Inc.; Yongdong Wang and Ming Gu,
James Beier, Bruker BioSpin; Professor F. X. Webster, CERNO Inc.; Jeff Okamitsu, Chemring Inc.
State University of New York College of Environmental Special thanks go to the late Professor Milton Orchin,
Science and Forestry; Pat Palumbo, Bill Strzynski, Lorne University of Cincinnati, for permission to use material
M. Fell, and Veronica Jackson, LECO Corporation; from his and the late H.H. Jaffe’s classic text, Theory and
Dr. Cedric Powell, Dr. William M. Haynes, Dr. Megan Applications of Ultraviolet Spectroscopy, Wiley, New York,
Harries, NIST; Jill Thomas and Michael Monko, Supelco; 1962. Long-term collaboration with Professor Paris D.N.
Michael Garriques and Karen Anspach, Phenomenex, Svoronos of QCC of City University of New York is
Inc.; A. Audino and Kerry Scoggins, SGE; Dr. Ronald acknowledged. The cheerful cooperation, patience,
Starcher, BURLE Electro-Optics, Inc.; Merrill Loechner, and invaluable assistance of Marie Scandone, Bio-Rad
Milestone, Inc.; Dr. Mike Collins, CEM Corporation; Informatics Division, in providing the Bio- Rad NMR,
xxvii
xxviiiAcknowledgments
UV, and IR spectra used in Chapters 3 through 5 and of introduced ICP- MS triple quadrupole instrument. Alan
Wes Rawlins, Cindy Addenbrook, Sean Battles, Dean Merrick, SPECTRO Analytical Instruments, provided
Llanas, Chris Wozniak, and Chris Lein, Sigma-Aldrich, the SPECTRO MS graphics. Dr. John F. Moulder, Physical
in providing the Aldrich NMR spectra in Chapter 3 and Electronics USA, Inc., provided many expert suggestions
the Supelco graphics in several chapters deserve a very, and revision of a large portion of Chapter 14, as well as
very special thank-you. Herk Alberry, Mike Farrell, and most of its graphics. Dr. Hans Joachim Schaefer and C.-
Danny Dirico of Albany Advanced Imaging, Albany, A. Schiller, ZAHNER-Elektrik GbmH & Co.KG, and
New York, are thanked for the MRI images and technical Professor Stephen P. Best, University of Melbourne,
assistance they provided for Chapter 3. To Dr. Christian Australia, provided graphics and examples for the discus-
Bock, Alfred- Wegner- Institute for Polar and Marine sion of spectroelectrochemistry. Dr. Chris Beasley, Gamry
Research, Bremerhaven, Germany, for the use of his MRI Instruments, Inc., contributed, reviewed, and provided
images in Chapter 3, Vielen Dank. Julie Powers, Toshiba the majority of graphics in the section on the quartz crystal
America Medical Systems, is gratefully acknowledged for microbalance in Chapter 15. Dr. Christin Choma, is thanked
the medical MRI system photos used in Chapter 3. Special for her invaluable help with understanding the applications
thanks go to Dr. Frank Dorman, Restek, for providing of microcalorimetry to the study of proteins.
their EZ- Chrom Chromatography Simulation Program, Thanks go to Irwin Schreiman, CambridgeSoft
used to create several of the figures in Chapter 12, and to Corporation, who provided the authors with a complete
Dr. Jack Cochran and Pam Decker, Restek, for invaluable copy of ChemDraw Ultra 12.0.
assistance. Dr. Z. Harry Xie, Bruker Optics, Inc., is grate- The authors wish to thank the following colleagues
fully acknowledged for providing the idea for and a large for their helpful comments and suggestions: Dr. O. David
portion of Section 3.9 on TD-NMR. Amy Herlihy, Agilent Sparkman, Pacific University; Professor Ronald Bailey,
Technologies, provided the information and example for Rensselaer Polytechnic Institute; Professor Peter Griffiths,
SWIFT NMR. Todd Strother, Thermo Fisher Scientific, University of Idaho; Dr. Elizabeth Williams; Professor
provided invaluable assistance with NIR in Chapter 4, both Julian Tyson, University of Massachusetts, Amherst.
with graphics and an excellent summary of chemometrics. Professor Emeritus Robert Gale, LSU, who coauthored
Timothy O. Deschaines, formerly with Thermo Fisher Chapter 15 for the fifth edition, is acknowledged for his sub-
Scientific, is thanked for his assistance with updating the stantive contributions to and review of that chapter.
Raman section, especially SERS. Harald Fischer, WITec The periodic table on the inside front cover is used with
GmbH, contributed the sections and provided graphics permission of Dragoset, R.A., Musgrove, A., Clark, C.W. and
for 3D confocal Raman imaging and true surface micros- Martin, W.C., Periodic Table: Atomic Properties of the
copy in Chapter 4. Thanks to Dr. Anne Lynn Gillian-Daniel, Elements, NIST SP 966 handout, August, 2019 (National
NMRFAM, University of Wisconsin, Madison (nmrfam. Institute of Standards and Technology, Gaithersburg, MD,
wisc.edu), for the 900 MHz NMR photos in Chapter 3. The www.nist.gov).
Bruker BioSpin website was invaluable for its EPR (ESR) Permission for the publication herein of Sadtler Spectra
tutorial, written by Dr. Ralph Weber. Dr. Weber contributed a has been granted by Bio- Rad Laboratories, Informatics
large portion of Section 6.12, with many helpful comments. Division. All Sadtler Spectra are Copyright Bio- Rad
Dr. Alexander Seyfarth, Bruker AXS, who significantly Laboratories, Informatics Division, Sadtler Software and
revised Chapter 8, is initiating interactive exercises to be Databases 2014. All rights reserved.
available online in the near future and is now the coauthor J.T.Baker® and ULTREX® are trademarks of Avantor
of that chapter. Dr. James R. White, Christopher J. White, Performance Materials, Inc.
and Colin T. Elliott, Active Spectrum, Inc., are thanked Last but certainly not least, Barbara Glunn Knott, senior
for their graphics and technical assistance with Micro-ESR. editor— analytical, industrial, and physical chemistry;
Dr. Andrzej Miziolek, US Army Research Laboratory, Alexis O’Brien, editor, Sherry Thomas, senior editorial
and Dr. Andrew Whitehouse, Applied Photonics Ltd., assistant, Fiona Macdonald, senior publisher, Jonathan
provided technical advice, literature, application notes, Pennell, cover design manager, and all of CRC Press/
and graphics for the section on LIBS. Drs. Doug Shrader Taylor & Francis Group, are thanked for their incredible
and Ed McCurdy, Agilent Technologies, provided the patience with and invaluable assistance to the authors.
graphics and examples for the description of their newly
Authors
Thomas J. Bruno was group leader in the Applied author of 250 professional papers, book chapters, and
Chemicals and Materials Division at NIST, Boulder, several books including Atomic Absorption Spectroscopy
Colorado, before retiring in 2019. He received his B.S. in and Atomic Spectroscopy, first and second editions.
chemistry from Polytechnic Institute of Brooklyn, and his He was editor in chief of Spectroscopy Letters and the
M.S. and Ph.D. in physical chemistry from Georgetown Journal of Environmental Science and Health (both
University. He first served as a National Academy of Marcel Dekker, Inc.); executive editor of Handbook
Sciences-National Research Council postdoctoral asso- of Spectroscopy Vol. 1 (1974), Vol. 2 (1974), Vol. 3
ciate at NIST. Dr. Bruno has researched fuels, explosives, (1981); and Practical Handbook of Spectroscopy (1991)
forensics, and environmental pollutants. He was also (all CRC Press). He served on the National University
involved in research on chemical separations, develop- Accreditation Committee from 1970– 1971. He was
ment of novel analytical methods, and novel detection a visiting distinguished professor at University of
devices for chromatography. Among his inventions are Colorado in 1972 and University of Sydney, Australia
the Advanced Distillation Curve method (for fuel char- in 1975. He served as the Gordon Conference Chairman
acterization), and PLOT-cryoadsorption (for vapor sam- in Analytical Chemistry in 1974. Professor Emeritus
pling). He has published over 270 research papers, eight James W. Robinson passed away in November, 2018, at
books, and has been awarded 10 patents. He serves as 95 years of age.
associate editor of the CRC Handbook of Chemistry
and Physics, and associate editor for Fuel Processing The late Eileen M. Skelly Frame was adjunct professor,
Technology. Bruno was awarded the Department of Department of Chemistry and Chemical Biology,
Commerce Bronze Medal in 1986 for his work “on the Rensselaer Polytechnic Institute (RPI), Troy, NY, and
thermophysics of reacting fluids”, and the Department head of Full Spectrum Analytical Consultants. Dr. Skelly
of Commerce Silver Medal in 2010 for “a new method Frame was the first woman commissioned from the
for analyzing complex fluid mixtures for introduction of Drexel University Army ROTC program. She graduated
new fuels into the U.S. energy infrastructure.” He was from Drexel summa cum laude in chemistry. She served
Distinguished Finalist for the 2011 CO-Labs Governor’s as Medical Service Corps officer in the U.S. Army from
Award for High Impact Research, and received the 1975 to 1986, rising to the rank of Captain. For the first
American Chemical Society Colorado Section Research five years of her military career, Dr. Skelly Frame was
Award in 2015. He has served as a forensic consultant stationed at the 10th Medical Laboratory at the U.S.
and/or an expert witness for the U.S. Department of Army Hospital in Landstuhl Germany. Thereafter, she
Justice (notably during the federal trial of Terry Nichols was selected to attend a three-year Ph.D. program in
for the Oklahoma City bombing), various United States Chemistry at Louisiana State University. She received her
Attorney’s offices, and various offices of the U.S. doctorate in 1982 and became the first female chemistry
Inspector General. He received a letter of commenda- professor at the U.S. Military Academy at West Point.
tion from the Department of Justice for these efforts in Following her military service, she joined the General
2002. He is currently working to develop science and Electric Corporation (now GE Global Research) and
technology education programs for the American judi- supervised the atomic spectroscopy laboratory. In add-
ciary. In this capacity he serves on the boards of the ition to her duties at RPI, she was Clinical and Adjunct
National Courts and Science Institute, and the Bryson Professor of Chemistry at Union College in Schenectady,
Institute for Judicial Education. In addition, he was NY. She was well known for her expertise in the use of
chairman of the Board of Trustees of the Mamie Doud instrumental analysis to characterize a wide variety of
Eisenhower Library for nine years. In his spare time, he substances, from biological samples and cosmetics to
is an avid and accomplished woodworker (and a less- high-temperature superconductors, polymers, metals,
than-accomplished welder and metal worker), and has and alloys. She was an active member of the American
hand-built all the furniture in his house. Chemical Society for 45 years, and a member of several
ASTM committees. Dr. Skelly Frame passed away in
The late James W. Robinson earned his B.S. (Hons), January of 2020.
Ph.D., and D.Sc. from the University of Birmingham,
England. He was professor emeritus of chemistry, George M. Frame II is a retired scientific director, Chemical
Louisiana State University, Baton Rouge, Louisiana. Biomonitoring Section of the Wadsworth Laboratory,
A fellow of the Royal Society of Chemistry, he is the New York State Department of Health, Albany. He has
xxix
newgenprepdf
xxxAuthors
a wide range of experience in analytical chemistry and member of the American Chemical Society. Dr. Frame
has worked at the GE Corporate R&D Center (now GE earned his AB in chemistry from Harvard College,
Global Research), Pfizer Central Research, the US Coast Cambridge, Massachusetts, and his Ph.D. in analytical
Guard R&D Center, the Maine Medical Center, and in chemistry from Rutgers University, New Brunswick,
the US Air Force Biomedical Sciences Corps. He is a New Jersey.
CHAPTER 1
Concepts of Instrumental Analytical Chemistry
1.1 INTRODUCTION: WHAT IS degrades in sunlight. These diverse applications make ana-
ANALYTICAL CHEMISTRY? lytical chemistry one of the broadest in scope of all sci-
entific disciplines. Analytical chemistry is critical to our
Perhaps the most functional definition of analytical understanding of biochemistry; medicinal chemistry; geo-
chemistry is that it is “the qualitative and quantitative char- chemistry; environmental science; atmospheric chemistry;
acterization of matter.” The word “characterization” is used the behavior of materials such as polymers, metal alloys,
in a very broad sense, and this is intentional because it can and ceramics; and many other scientific disciplines.
encompass material properties as well as composition. It For many years, analytical chemistry relied on chem-
may mean the identification of the chemical compounds or ical reactions to identify and determine the components pre-
elements present in a sample to answer questions such as sent in a sample. These types of classical methods, often
“Is there any vitamin E in this shampoo as indicated on the called “wet chemical methods,” usually required that a part
label?” or “Is this white tablet an aspirin tablet?” or “Is this of the sample be taken and dissolved in a suitable solvent
piece of metal iron or nickel?” This type of characteriza- if necessary and the desired reaction carried out. The most
tion, to tell us what is present, is called qualitative analysis. important analytical fields based on this approach were
Qualitative analysis is the identification of one or more volumetric and gravimetric analyses. Acid–base titrations,
chemical species present in a material. Characterization oxidation–reduction titrations, and gravimetric determin-
may also mean the determination of how much of a par- ations, such as the determination of silver by precipitation
ticular compound or element is present in a sample, to as silver chloride, are all examples of wet chemical ana-
answer questions such as “How much acetylsalicylic acid is lyses. These types of analyses require a high degree of skill
in this aspirin tablet?” or “How much nickel is in this steel?” and attention to detail on the part of the analyst if accurate
This determination of how much of a species is present in and precise results are to be obtained. They are also time
a sample is called quantitative analysis. Quantitative ana- consuming, and the demands of today’s high-throughput
lysis is the determination of the exact amount of a chem- pharmaceutical development labs, forensic labs, com-
ical species present in a sample. The chemical species may mercial environmental labs, and industrial quality control
be an element, compound, or ion. The compound may be labs often do not permit the use of such time-consuming
organic or inorganic. Characterization can refer to the entire methods for routine analysis. In addition, it may be neces-
sample (bulk analysis), such as the elemental composition sary to analyze samples without destroying them. Examples
of a piece of steel, or to the surface of a sample (surface include evaluation of valuable artwork to determine if a
analysis), such as the identification of the composition and painting really is by a famous “old master” or is a modern
thickness of the oxide layer that forms on the surface of forgery, as well as in forensic analysis, where the evidence
most metals exposed to air and water. The characterization may need to be preserved. For these types of analyses, non-
of a material may go beyond chemical analysis to include destructive analysis methods are needed, and wet chem-
structural determination of materials, the measurement of ical analysis will not do the job. Wet chemical analysis is
physical properties of a material, and the measurement still used in specialized areas of analysis, and is sometimes
of physicochemical parameters such as reaction kinetics. cost effective, but many of the volumetric methods have
Indeed, to the extent that properties depend on the chemical been transferred to automated instruments. Classical ana-
identity, it can be argued that all properties of matter are the lysis and instrumental analysis are similar in many respects,
result of the chemical composition. such as in the need for proper sampling, sample prepar-
Examples of such physicochemical measurements are ation, assessment of accuracy and precision, and proper
the degree to which a polymer is crystalline as opposed to record keeping. Some of the topics discussed briefly in this
amorphous, the temperature at which a material loses its chapter are covered at greater length in more general texts
water of hydration, how long it takes for antacid “brand on analytical chemistry and quantitative analysis. Several of
A” to neutralize stomach acid, and how fast a pesticide these types of texts are listed in the bibliography; the book
DOI: 10.1201/9781003188544-1 1
2 Undergraduate Instrumental Analysis
by Bruno and Svoronos has a comprehensive treatment Many analytical results are expressed as the concen-
including flow charts. tration of the measured substance in a certain amount of
Most analyses today are carried out with specially sample. The measured substance is called the analyte.
designed electronic instruments controlled by computers. Commonly used concentration units include molarity
These instruments make use of the interaction of electro- (moles of substance per liter of solution), mass percent
magnetic radiation and matter, or of some physical property (grams of substance per gram of sample × 100%), and units
of matter, to characterize the sample being analyzed. Often for trace levels of substances. One part per million (ppm)
these instruments have automated sample introduction, by mass is one microgram of analyte in a gram of sample,
data processing, and even sample preparation. To under- that is, 1 × 10−6 g analyte/g sample. One part per billion
stand how the instrumentation operates and what infor- (ppb) by mass is one nanogram of analyte in a gram of
mation it can provide requires knowledge of chemistry, sample or 1 × 10−9 g analyte/g sample. For many elements,
physics, mathematics, and engineering. The fundamentals the technique known as inductively coupled plasma mass
of common analytical instruments and how measurements spectrometry (ICP-MS) can detect parts per trillion of the
are performed with these instruments are the subjects of element, that is, picograms of element per gram of sample
the following chapters on specific instrumental techniques. (1 × 10−12 g analyte/g sample). To give you a feeling for
The analytical chemist must not only know and understand these quantities, a million seconds is ∼12 days (11.57 days,
analytical chemistry and instrumentation but must also be to be exact). One part per million in units of seconds would
able to serve as a problem solver to colleagues in other sci- be 1 s in 12 days. A part per billion in units of seconds
entific areas. This means that the analytical chemist may would be 1 s in ∼32 years, and one part per trillion is 1 s in
need to understand materials science, metallurgy, biology, 32,000 years. Today, lawmakers set environmental levels of
pharmacology, agricultural science, food science, geology, allowed chemicals in air and water based on measurements
and other fields. The field of analytical chemistry is advan- of compounds and elements at part per trillion levels
cing rapidly. To keep up with the advances, the analytical because instrumental methods can detect part per trillion
chemist must understand the fundamentals of common ana- levels of analytes. It is the analytical chemist who is respon-
lytical techniques and their capabilities and shortcomings. sible for generating the data that these lawmakers rely on.
The analytical chemist must understand the problem to be A table of commonly encountered constants, multiplication
solved, select the appropriate technique or techniques to be factors, and their prefixes is found inside the textbook cover
used, design the analytical experiment to provide relevant (a comprehensive listing can be found in the book by Bruno
data, and ensure that the data obtained are valid. Merely and Svoronos). The student should become familiar with
providing data to other scientists is not enough; the analyt- these prefixes, since they will be used throughout the text.
ical chemist must be able to interpret the data and commu-
nicate the meaning of the results, together with the accuracy 1.1.1 The Difference between Weight and Mass
and precision (the reliability) of the data, to scientists who
will use the data. In addition to understanding the scien- The terms weight and mass are often used interchange-
tific problem, the modern analytical chemist often must also ably, and that is usually fine on planet Earth. Mass is the
consider factors such as time limitations and cost limitations amount of substance (an inertial property, the sum of all the
in providing an analysis. Whether one is working for a gov- electrons, protons and neutrons) in an object, while weight
ernment regulatory agency, a hospital, a private company, is the gravitational interaction between objects that have
or a university, analytical data must be legally defensible. mass. A (stationary) electronic balance measures the gravi-
It must be of known, documented quality. Record keeping, tational force of an object (i.e., its weight) by use of a load
especially computer record keeping; assessing accuracy cell, and converts it to mass mathematically (weight =mass
and precision; statistical handling of data; documenting; × gravitation, W =mg, where g is roughly 9.81 N/kg). Use
and ensuring that the data meet the applicable technical that same balance on Mars in the rover and you will not get
standards are especially critical aspects of the job of modern the correct mass. Use that same balance in a moving ele-
analytical chemists. vator and, likewise, you will not get the correct mass. Some
Analytical chemistry uses many specialized terms that highly precise measurements require the measurement
may be new to you. The definitions of these terms, usu- of g locally to obtain a correction factor, but for analyt-
ally shown in boldface, must be learned. The units used ical measurements (and especially for weighing by diffe-
in this text are, for the most part, the units of the Système rence) the conversion to mass used by electronic balances is
International d’Unités (SI system). The SI system is used adequate. Buoyancy corrections were commonly required
around the world by scientists and engineers. The tables when two pan analytical balances were common (and are
inside the textbook covers give the primary units of meas- still required when weighing large objects such as gas
urement in the SI system. A comprehensive list of SI units, cylinders), but for electronic balances, this consideration is
SI-derived units and definitions, as well as non-SI units typically only required for a calibration. So, what does this
may be found at the US National Institute of Standards and mean for our analytical measurements? It means that our
Technology (NIST) website at http://physics.nist.gov. balance will measure weight, and provide us with mass. It is
Concepts of Instrumental Analytical Chemistry3
therefore more correct to say: mass. And while we are on the 1.2.1 Defining the Problem
topic, remember that the instrument that you use to measure
the weight and read out the mass is called a balance, not a The analytical chemist must find out what informa-
scale. Scientists use a balance; butchers use a scale. tion needs to be known about the sample, material, or
process being studied, how accurate and precise the ana-
lytical information must be, how much material or sample
1.2 ANALYTICAL APPROACH is available for study, and if the sample must be analyzed
without destroying it. Is the sample organic or inorganic?
A major personal care products manufacturer receives Is it a pure material or a mixture? Does the customer want
a phone call from an outraged customer whose hair has a bulk analysis or information about a particular fraction of
turned green after using their “new, improved shampoo.” the sample, such as the surface? Does the customer need
The US Coast Guard arrives at the scene of an oil spill in to know if the sample is homogeneous or heterogeneous
a harbor and finds two ship’s captains blaming each other with respect to a given analyte? Does the customer need
for the spill. A plastics company that sells bottles to a elemental information or information about the chemical
water company bottling “pure, crystal-clear spring water” species (ionic or molecular, particular oxidation states)
discovers that the 100,000 new empty bottles it is ready present in the sample? The answers to such questions will
to ship are slightly yellow in color instead of crystal clear. guide the analyst in choosing the analytical method. Of
A new, contagious disease breaks out, and people are dying course, sometimes the answers to some of the questions
of flu-like symptoms. What caused the problem? How can may be part of the problem. If the sample is an unknown
it be prevented in the future? Who is at fault? Can a vaccine material, the analyst must find out if it is organic or inor-
or drug treatment be developed quickly? These sorts of ganic, pure or a mixture, as part of solving the problem.
problems and many more occur daily around the world, in The analyte is the substance to be measured; everything
industry, in medicine, and in the environment. A key figure else in the sample is called the matrix. Of course, there
in the solution of these types of problems is the analyt- may be more than one analyte in a given sample. The terms
ical chemist. The analytical chemist is first and foremost analysis and analyze are applied to the sample under study,
a problem solver and, to do that, must understand the ana- as in “this water was analyzed for nitrate ion” or “an ana-
lytical approach, the fundamentals of common analytical lysis of the contaminated soil was performed.” Water and
techniques, their uses, and their limitations. soil are the samples being analyzed. The terms determine
The approach used by analytical chemists to solve and determination are applied to the measurement of the
problems may include the following steps: analyte in the sample, as in “nitrate ion was determined in
the water sample,” “a determination of lead in blood was
1. Defining the problem and designing the analytical made because the symptoms indicated lead poisoning,” or
method. “an analysis of the soil was performed and cyanide levels
2. Sampling and sample storage.
were determined.” Nitrate ion, lead, and cyanide are the
3. Sample preparation.
4. Performing the measurement.
analytes being determined. Other components in the sample
5. Assessing the data and the corresponding uncertainty. matrix may interfere with the measurement of the analyte;
6. Method validation. such components are called interferences.
7. Documentation. A sample may be homogeneous, that is, it has the same
chemical composition everywhere within the sample. Plain
General sample preparation will be discussed in this vanilla ice cream, a milk chocolate bar, and salt water are
chapter, but instrument- specific sample preparation is examples of homogeneous materials. Many samples are
included in the appropriate chapter on each technique. heterogeneous; the composition varies from region to
Method validation and documentation will not be covered region within the sample. Vanilla ice cream with raisins in
as the focus of this text is on instrumentation. The text by it and a chocolate bar with whole almonds in it are hetero-
Christian cited in the bibliography has an excellent intro- geneous; you can see the composition difference. In most
duction to validation and documentation for the interested real samples, the heterogeneity may not be visible to the
student. human eye. The variation in composition can be random,
Although the steps in solving analytical problems usu- or it can be segregated into regions of distinctly different
ally follow the listed order, knowledge of basic statistics compositions.
is useful not just for handling the data and method valid- A significant part of defining the problem is the decision
ation but is required for proper sampling and selection of between performing a qualitative analysis and a quantitative
an analytical method. The statistics and definitions needed analysis. Often the problem is first tackled with a qualita-
to understand what is meant by accuracy, precision, tive analysis, followed by a quantitative analysis for specific
error, and so on are covered in Section 1.3. Steps 1 and analytes. The analyst needs to communicate with the cus-
2 are covered in this section, while steps 3 through 5 are tomer who is requesting the analysis. Two-way communica-
discussed in the sections following Section 1.3. tion is important, to be certain that the problem to be solved
is understood and to be sure that the customer understands all the elements in the periodic table, even when only trace
the capabilities and limitations of the analysis. amounts are present, but often require that the sample be
in the form of a solution. If the sample is a rock or a piece
1.2.1.1 Qualitative Analysis of glass or a piece of biological tissue, the sample usually
must be dissolved in some way to provide a solution for
Qualitative analysis is the branch of analytical chem- analysis. We will see how this is done later in this chapter.
istry that is concerned with questions such as “What makes The analyst can determine accurately what elements are
this water smell bad?” “Is there gold in this rock sample?” present, but information about the molecules in the sample
“Is this sparkling stone a diamond or cubic zirconia?” “Is is often lost in the sample preparation process. The advan-
this plastic item made of polyvinyl chloride, polyethylene, tage of ICP-OES and ICP-MS is that they are very sensitive;
or polycarbonate?” “What is this white powder?” concentrations at or below 1 ppb of most elements can be
Some methods for qualitative analysis are nondestruc- detected using these methods.
tive, that is, they provide information about what is in the If the sample is organic, that is, composed primarily
sample without destroying the sample. These are often the of carbon and hydrogen, qualitative analysis can provide
best techniques to begin with, because the sample can be chemical and structural information to permit identifica-
used for subsequent analyses if necessary. To identify what tion of the compound. The IR spectrum will provide iden-
elements are present in a sample nondestructively, a qualita- tification of the class of compound, for example, ketone,
tive elemental analysis method such as X-ray fluorescence acid, or ether. NMR spectroscopy and mass spectrometry
(XRF) spectroscopy can be used. Modern XRF instruments, (MS), as we shall see in the appropriate chapters, provide
discussed in Chapter 9, can identify all elements from detailed structural information, often including the relative
sodium to uranium, and some instruments can measure molecular mass (RMM, or MW) of the compound. Use of
elements from beryllium to uranium. The sample is usually IR, NMR, and MS, combined with quantitative elemental
not harmed by XRF analysis. For example, XRF could easily analysis to accurately determine the percentage of carbon,
distinguish a diamond from cubic zirconia. Diamond is, of hydrogen, oxygen, and other elements, is the usual process
course, a crystalline form of carbon; most XRF instruments by which analytical chemists identify organic compounds.
would see no elemental signal from the carbon in a diamond This approach is required to identify new compounds
but would see a strong signal from the element zirconium synthesized by pharmaceutical chemists, for example. In a
in cubic zirconia, a crystalline compound of zirconium and simple example, elemental analysis of an unknown organic
oxygen. Qualitative molecular analysis will tell us what compound might provide an empirical formula of C2H5. An
molecules are present in a material. The nondestructive empirical formula is the simplest whole number ratio of the
identification of molecular compounds present in a sample atoms of each element present in a molecule. For any given
can often be accomplished by the use of nuclear magnetic compound, the empirical formula may or may not coincide
resonance (NMR) spectroscopy, discussed in Chapter 6, or with the molecular formula. A molecular formula contains
by infrared (IR) spectroscopy, discussed in Chapter 5. IR the total number of atoms of each element in a single mol-
spectroscopy can provide information about organic func- ecule of the compound. The results from IR, NMR, and MS
tional groups present in samples, such as alcohols, ketones, might lead the analytical chemist to the molecular formula
carboxylic acids, amines, and thioethers. If the sample is C4H10 and would, in addition, indicate which of the following
a pure compound such as acetylsalicylic acid (the active two different structures shown our sample was:
ingredient in aspirin), the IR spectrum may be able to iden-
tify the compound exactly, because the IR spectrum for a H
compound is unique, like a fingerprint. Qualitative iden- |
CH3−CH2−CH2−CH3 H3C−C−CH3
tification of polymers for recycling can be done using IR |
spectroscopy, for example. NMR gives us detailed infor- CH3
mation about the types of protons, carbon, and other atoms
n–Butane 2-Methyl-propane (or iso-butane)
in organic compounds and how the atoms are connected.
NMR can provide the chemical structure of a compound
without destroying it. These two structures are two different compounds with
Many methods used for qualitative analysis are destruc- the same molecular formula. They are called isomers.
tive; either the sample is consumed during the analysis or Elemental analysis cannot distinguish between these
must be chemically altered in order to be analyzed. The most isomers, but NMR and MS usually can distinguish isomers.
sensitive and comprehensive elemental analysis methods Another example of a more difficult qualitative analysis
for inorganic analysis are ICP atomic emission spectrom- problem is the case of the simple sugar, erythrose. The
etry (ICP-AES or ICP optical emission spectrometry [ICP- empirical formula determined by elemental analysis is
OES]), discussed in Chapter 8, and ICP-MS, discussed in CH2O. The molecular formula, C4H8O4, and some of the
Chapters 10 and 11. These techniques can identify almost structure can be obtained from IR, NMR, and MS, but we
Mirror plane
CHO CHO
H C OH HO C H
H C OH HO C H
CH2OH CH2OH
D-(–)-Erythrose L-(+)-Erythrose
Figure 1.2 Enantiomers of glyceraldehyde.
Figure 1.1 Isomers of erythrose.

relationship between the D and L configuration and the
cannot tell from these techniques which of the two possible direction of rotation of plane-polarized light. Figure 1.2
isomers shown in Figure 1.1 is our sample. shows the simplest sugar, glyceraldehyde. It also has two
These two erythrose molecules are chiral, that is, enantiomers, one D and one L, but the D enantiomer of
they are nonsuperimposable mirror-image isomers, called glyceraldehyde rotates light in the opposite direction from
enantiomers. Imagine sliding the molecule on the left in D-erythrose.
the plane of the paper, through the “mirror plane” indicated If organic compounds occur in mixtures, separation
by the arrow, over the molecule on the right; the OH groups of the mixture often must be done before the individual
will not be on top of each other. Imagine turning the left components can be identified. Techniques such as gas
molecule in the plane of the paper upside down and then chromatography (GC), liquid chromatography (LC), and
sliding it to the right; now the OH groups are lined up, capillary electrophoresis (CE) are often used to separate
but the CHO and CH2OH groups are not. That is what is mixtures of organic compounds prior to identification of the
meant by nonsuperimposable. You can do whatever you like components. These methods are discussed in Chapters 12
to the two molecules except remove them from the plane through 14.
of the paper; no matter how you move them, they will not Table 1.1 lists some common commercially available
be superimposable. They have the same molecular formula instrumental methods of analysis and summarizes their
(C4H8O4); the same IR spectrum, mass spectrum, and usefulness for qualitative elemental or molecular ana-
NMR spectrum; and many of the same physical properties lysis. Table 1.2 gives a very brief summary of the use of
such as boiling point and refractive index (RI). Such chiral the methods. Analyte concentrations that can be determined
compounds can be distinguished from each other by inter- by common methods of instrumental analysis are presented
action with something else that possesses chirality or by in Table 1.3. The concentration of analyte that can be
interaction with plane-polarized light. Chiral compounds determined in real samples will depend on the sample and
will interact differently with other chiral molecules, and this on the instrument, but Table 1.3 gives some indication of the
interaction forms the basis of chiral chromatography. Chiral sensitivity and working range of methods.
chromatography (Chapter 14) can be used to separate the
two erythrose compounds shown. Chiral compounds also 1.2.1.2 Quantitative Analysis
differ in their behavior toward plane-polarized light, and the
technique of polarimetry can be used to distinguish them. When qualitative analysis is completed, the next
One of the erythrose enantiomers rotates plane-polarized question is often “How much of each or any component
light to the right (clockwise); this compound is dextrorota- is present?” or “Exactly how much gold is this rock?” or
tory and is given the symbol (+) as part of its name. The “How much of the organochlorine pesticide dieldrin is
other enantiomer rotates the plane of polarization to the left in this drinking water?” The determination of how much
(counterclockwise); this compound is levorotatory and is is quantitative analysis. Analytical chemists express how
given the symbol (−) in its name. Such compounds are said much in a variety of ways, but often in terms of concentra-
to be optically active. Chiral compounds are very important tion, the amount of analyte in a given amount of sample.
because biochemical reactions are selective for only one of Concentration is an expression of the quantity of analyte in
the two structures and only one of the two enantiomers is a given volume or mass of sample. Common concentration
biologically active. Biochemists, pharmaceutical chemists, units include molarity, defined as moles of analyte per liter
and medicinal chemists are very interested in the identifica- of sample and symbolized by M or mol/L; percent by mass,
tion, synthesis, and separation of only the biologically defined as grams of analyte per gram of sample × 100 %,
active compound. The letters D and L in the name of symbolized as % or % (mass/mass); and parts per million,
the sugar refer to the position of the alcohol group on the defined as micrograms of analyte per gram of sample (ppm,
carbon closest to the bottom primary alcohol. There is no μg/g). For dilute aqueous solutions, 1 mL of solution has
Table 1.1 Instrumental Methods of Analysis
Qualitative Quantitative
Method Elemental Molecular Elemental Molecular
Atomic absorption spectrometry No No Yes No

Atomic emission spectrometry Yes No Yes No
Capillary electrophoresis Yes Yes Yes Yes
Electrochemistry Yes Yes Yes Yes
Gas chromatography No Yes No Yes
ICP-mass spectrometry Yes No Yes No
Infrared spectroscopy No Yes No Yes
Ion chromatography Yes Yes Yes Yes
Liquid chromatography No Yes No Yes
Mass spectrometry Yes Yes Yes Yes
Nuclear magnetic resonance No Yes No Yes
Raman spectroscopy No Yes No Yes
Thermal analysis No Yes No Yes
UV/VIS spectrophotometry Yes Yes Yes Yes
UV absorption No Yes No Yes
UV fluorescence No Yes No Yes
X-ray absorption Yes No Yes No
X-ray diffraction No Yes No Yes
X-ray fluorescence Yes No Yes No
Table 1.2 Principal Applications of Instrumental Methods of Analysis
Molecular Analysis
Nuclear magnetic resonance (NMR) spectroscopy

Qualitative analysis: NMR is one of the most powerful methods available for determining the structure of molecules. It identifies
the number and type of protons and carbon atoms in organic molecules, for example, it distinguishes among aromatic, aliphatic,
alcohols, and aldehydes. Most importantly, it also reveals the positions of the nuclei in the molecule relative to each other.
For example, NMR will distinguish between CH3—CH2—CH2OH and CH3—CHOH—CH3. It does not provide the RMM of the
compound. NMR is also applied to compounds containing heteroatoms such as sulfur, nitrogen, fluorine, phosphorus, and silicon.
Quantitative analysis: NMR is useful at % concentration levels, but trace levels (ppm) are becoming attainable with reasonable
accuracy.
Infrared (IR) spectroscopy and Raman spectroscopy
Qualitative analysis: IR readily identifies organic functional groups present in molecules including groups containing heteroatoms—
O, S, N, Si, halides. The IR spectrum is a fingerprint for a given compound, making it a very useful qualitative method. Classical
mid-IR spectroscopy cannot be done on aqueous solutions. It does not give the RMM of the compound. Raman spectroscopy
complements IR spectroscopy and is useful for aqueous samples.
Quantitative analysis: IR is used routinely for the quantitative analysis of organic compounds, particularly at % concentration levels. It
is used for liquid, solid, and gaseous samples. The related field of Raman spectroscopy complements IR.
Ultraviolet (UV) absorption spectroscopy
Qualitative analysis: UV absorption can be used for identifying functional groups and the structures of molecules containing
unsaturated bonds (π electrons), such as
H H H H
C C C C
H H
Table 1.2 (Continued) Principal Applications of Instrumental Methods of Analysis

and aromatics and lone-pair electrons, such as those in pyridine:
N
lone pair
It does not indicate RMM or give useful information on saturated bonds (σ bonds). NMR and IR have almost entirely replaced UV
absorption spectroscopy for organic compound identification.
Quantitative analysis: UV absorption is used routinely for the quantitative determination of unsaturated compounds such as those
found in natural products. The method is subject to spectral overlap and therefore interference from other compounds in the
sample.
UV fluorescence
Qualitative analysis: UV fluorescence is used for the determination of unsaturated compounds, particularly aromatics. It does not
indicate RMM but gives some indication of the functional groups present. It is much more sensitive than UV absorption.
Quantitative analysis: UV fluorescence is a very sensitive method of analysis (10−8 g/g or 10 ppb), but it is subject to many kinds of
interference, both from quenching effects and from spectral overlap from other compounds.
UV and visible (UV/VIS) spectrophotometry
Qualitative analysis: Organic or inorganic reagents are used for specific tests for many elements or compounds by forming a
compound that absorbs at specific wavelengths. The products may or may not be colored. If the compounds are colored, analysis
may be carried out visually (colorimetric analysis by eye), but the use of a spectrometer is more accurate.
Quantitative analysis: Sensitive and selective methods have been developed for most elements and many functional groups. It is used
extensively in routine analysis of water, food, beverages, industrial products, etc.
X-ray diffraction (XRD)
Qualitative analysis: XRD is used for the measurement of crystal lattice dimensions and to identify the structure and composition of all
types of crystalline inorganic and organic materials.
Quantitative analysis: XRD is used for the determination of percent crystallinity in polymers, the composition of mixtures, mixed
crystals, soils, and natural products.
X-ray absorption spectroscopy
Qualitative analysis: X-ray absorption reveals the contours and location of high atomic mass elements in the presence of low atomic
mass matrices or holes in the interior of solid samples (voids). Examples are bone locations in the human body, the contents of
closed suitcases, old paintings hidden under new painting on a canvas, and voids in welded joints and opaque solid objects.
Organic mass spectrometry (MS)
Qualitative analysis: MS can be used to identify the RMM of organic and inorganic compounds, from very small molecules to
large polymers and biological molecules (>100,000 Da). MS is a powerful tool in the determination of the structure of organic
compounds. Fragmentation patterns can reveal the presence of substructure units within the molecule.
Quantitative analysis: MS is used extensively for the quantitative determination of the organic components of solid, liquid, and gas
samples.
Thermal analysis (TA)
Qualitative analysis: TA is used to identify inorganic and some organic compounds using very small quantities of sample. It is also
used to identify phase changes, chemical changes on heating, heats of fusion, melting points, boiling points, drying processes,
decomposition processes, and the purity of compounds.
Quantitative analysis: TA can be used for the quantitative determination of the components of an inorganic sample, particularly at high
concentration levels.
Gas chromatography (GC)
Qualitative analysis: GC can be used to separate the components of complex mixtures of gases or of volatile compounds. By
comparison with known standards, it can identify components based on retention time.
Quantitative analysis: GC is an accurate method for quantitative analysis based on the area of the peak and comparison with
standards. It is used extensively in organic, environmental, clinical, and industrial analysis. GC with MS detection (GC-MS) is a
routine and powerful tool for quantitative analysis of organic compounds in environmental and biological samples.
(continued)
Table 1.2 (Continued) Principal Applications of Instrumental Methods of Analysis
Liquid chromatography (LC, high-performance LC [HPLC])

Qualitative analysis: LC is used for the identification of components of liquid mixtures, including polar compounds, ions, high-MW
components, and thermally unstable compounds. Identification is based on retention time and comparison with standards.
Quantitative analysis: LC is used for the quantitative determination of components in mixtures, especially for high-MW or thermally
unstable compounds. It is particularly useful for separating complicated mixtures such as natural products derived from plants or
animals and biological samples such as urine and blood. Ion chromatography (IC) is used routinely in water analysis. LC with MS
detection (LC-MS) is a routine and powerful tool for quantitative analysis of organic compounds in environmental and biological
samples.
Capillary electrophoresis (CE)
Qualitative analysis: Used for the separation and identification of ions and neutral molecules in mixtures. Can be used for ions in
aqueous solution and for organic ions.
Quantitative analysis: Quantitative determination of ions can be accomplished following separation, as in IC and LC.
Elemental analysis
Atomic emission spectrometry (AES), optical emission spectrometry (OES)
Qualitative analysis: AES is an almost comprehensive method for qualitative elemental analysis for metals, metalloids, and nonmetals
with the exception of some of the permanent gases. Its sensitivity range is great, varying from ppb to percent levels. Many elements
can be detected simultaneously. Spectral overlap is the major limitation.
Quantitative analysis: AES is used extensively for the quantitative determination of elements in concentrations from percent levels
down to ppb. Liquids, slurries, and solids can be analyzed using the appropriate instrumentation.
Flame photometry (flame atomic emission spectrometry)
Qualitative analysis: Flame photometry is particularly useful for the determination of alkali metals and alkaline-earth metals. It
provides the basis for flame tests used in qualitative analysis schemes.
Quantitative analysis: Flame photometry is used for the quantitative determination of alkali metals and alkaline-earth metals in blood,
serum, and urine in clinical laboratories. It provides much simpler spectra than those found in other types of AES, but its sensitivity
is much reduced.
Atomic absorption spectrometry (AAS)
Qualitative analysis: AAS is not used routinely for qualitative analysis, since with most instruments, it is only possible to test for one
element at a time.
Quantitative analysis: AAS is a very accurate and sensitive method for the quantitative determination of metals and metalloids down
to absolute amounts as low as picograms for some elements. It cannot be used directly for the determination of nonmetals.
X-ray fluorescence (XRF)
Qualitative analysis: XRF is useful for elements with atomic numbers greater than 4, including metals and nonmetals. For qualitative
analysis, no sample preparation is required, and the method is generally nondestructive.
Quantitative analysis: XRF is used extensively for quantitative determination of elements in alloys and mineral samples, particularly of
elements with high atomic masses. Sample preparation is complex for quantitative analysis.
Inorganic mass spectrometry (MS)
Qualitative analysis: Inorganic MS can identify elements, isotopes, and polyatomic ions in solutions and solid samples.
Quantitative analysis: Inorganic MS can determine elements at ppt concentrations or below. It is used for simultaneous multielement
analysis for metals and nonmetals. Inorganic MS provides the isotope distribution of the elements. Special mass spectrometers are
used for accurate isotope ratio measurements used in geology and geochemistry.
a mass of 1 g (because the density of water is 1 g/mL), color produced (colorimetry). Using these methods, it was
so solution concentrations are often expressed in terms of found, for example, that dry sodium chloride, NaCl, always
volume. A part per million of analyte in dilute aqueous solu- contained 39.33% Na and 60.67% Cl. The atomic theory
tion is equal to one microgram per milliliter of solution (μg/ was founded on early quantitative results such as this, as
mL), for example. were the concept of valence and the determination of atomic
The first quantitative analytical fields to be developed masses. Today, quantitative inorganic elemental analysis is
were for quantitative elemental analysis, which revealed performed by atomic absorption spectrometry (AAS), AES
how much of each element was present in a sample. of many sorts, inorganic MS (such as ICP-MS), XRF, ion
These early techniques were not instrumental methods, chromatography (IC), and other techniques discussed in
for the most part, but relied on chemical reactions, phys- detail in later chapters.
ical separations, and weighing of products (gravimetry), In a similar fashion, quantitative elemental analysis for
titrations (titrimetry or volumetric analysis), or production carbon, hydrogen, nitrogen, and oxygen enabled the chemist
of colored products with visual estimation of the amount of to determine the empirical formulas of organic compounds.
Table 1.3 Analytical Concentration Ranges for Common Instrumental Methods
Ultratrace
Technique Destructive (<1 ppm) Trace (1 ppm–0.1%) Minor (0.1%–10%) Major (>10%)
X-ray diffraction No No No Yes Yes

Nuclear magnetic resonance No No Yes Yes Yes
X-ray fluorescence No No Yes Yes Yes
Infrared spectroscopy No No Yes Yes Yes
Raman spectroscopy No No Yes Yes Yes
UV/VIS spectrometry No No Yes Yes Yes
Colorimetry No Yes Yes Yes No
Molecular fluorescence spectrometry No Yes Yes Yes Yes
Atomic absorption spectrometry Yes Yes Yes Yes No
Atomic emission spectrometry Yes Yes Yes Yes Yes
Atomic fluorescence spectrometry Yes Yes Yes No No
ICP-mass spectrometry Yes Yes Yes Yes No
Organic mass spectrometry Yes Yes Yes Yes Yes
GC-MS Yes Yes Yes Yes Yes
LC-MS Yes Yes Yes Yes Yes
Potentiometry No Yes Yes Yes Yes
Voltammetry No Yes Yes Yes Yes
Gas chromatography May be Yes Yes Yes Yes
High-performance liquid May be Yes Yes Yes Yes
chromatography
Ion chromatography May be Yes Yes Yes Yes
Capillary electrophoresis No Yes Yes Yes Yes
Thermal analysis May be No No Yes Yes
Note: The destructive nature of the instrumental method is characterized. A sample may be destroyed by a nondestructive instrumental
method, depending on the sample preparation required. The chromatographic techniques may be destructive or nondestructive,
depending on the type of detector employed. The nondestructive detectors generally limit sensitivity to “trace.” Molecular fluorescence
is not destructive if the molecule is inherently fluorescent. It may be if the molecule requires derivatization. A method with “yes” for
ultratrace and “no” for major concentrations reflects the linear working range. Such methods can measure “majors” if the sample is
diluted sufficiently.
An empirical formula is the simplest whole number ratio CO2 and all of the hydrogen to H2O. The CO2 and H2O
of the atoms of each element present in a molecule. For were collected and weighed, or the volume of the gas was
any given compound, the empirical formula may or may determined by displacement of liquid in a measuring device.
not coincide with the molecular formula. A molecular for- To distinguish between butane (C4H10), which contains
mula contains the total number of atoms of each element 82.76% C and 17.24% H, and pentane (C5H12), which contains
in a single molecule of the compound. For example, 83.33% C and 16.66% H, required great skill using manual
ethylene and cyclohexane have the same empirical formula, combustion analysis. Today, automated analyzers based on
CH2, but molecular formulae of C2H4 and C6H12, respect- combustion are used for quantitative elemental analysis for
ively. The empirical formula of many sugars is CH2O, but C, H, N, O, S, and the halogens in organic compounds. These
the molecular formulae differ greatly. The molecular for- analyzers measure the evolved species by GC, IR, or other
mula of glucose is C6H12O6, fructose is C6H12O6, erythrose techniques. These automated analyzers require only micro-
is C4H8O4, and glyceraldehyde is C3H6O3. An example of gram amounts of sample and a few minutes to provide data
a molecule whose empirical formula is the same as the and empirical formulas that used to take hours of skilled
molecular formula is tetrahydrofuran (THF), an important analytical work. Quantitative elemental analysis cannot dis-
organic solvent. The molecular formula for THF is C4H8O; tinguish between isomers, which are compounds with the
there is only one oxygen atom, so there can be no smaller same molecular formula but different structures. Glucose
whole number ratio of the atoms. Therefore, C4H8O is also and fructose have the same molecular formula, but glucose
the empirical formula of THF. is a sugar with an aldehyde group in its structure, while fruc-
Empirical formulas of organic compounds were derived tose is a sugar with a ketone group in its structure. They
mainly from combustion analysis, where the organic com- cannot be distinguished by elemental analysis but are easily
pound is heated in oxygen to convert all of the carbon to distinguished by their IR and NMR spectra.
Quantitative molecular analysis has become increas- is that they are “green chemistry” processes, that is, that
ingly important as the fields of environmental science, the solvents used are of low toxicity or biodegradable, that
polymer chemistry, biochemistry, pharmaceutical chem- waste is minimized, and that chemicals used in the analysis
istry, natural products chemistry, and medicinal chemistry are recycled when possible.
have grown. Techniques such as GC, LC, or HPLC, CE, Designing a good analytical method requires knowing
MS, fluorescence spectrometry, IR, and XRD are used to how to obtain a representative sample of the material to be
determine the amount of specific compounds, either pure analyzed, how to store or preserve the sample until analysis,
or in mixtures. These techniques have become highly and how to prepare the sample for analysis. The analyst must
automated and extremely sensitive, so that only micrograms also know how to evaluate possible interferences and errors
or milligrams of sample are needed in most cases. The chro- in the analysis and how to assess the accuracy and precision
matography techniques, which can separate mixtures, have of the analysis. These topics will be discussed subsequently,
been “coupled” to techniques like MS, which can iden- and specific interferences for given instrumental methods
tify and quantitatively measure the components in a mix- are discussed in the following chapters.
ture. Such techniques, like GC-MS and LC-MS, are called There are many analytical procedures and methods
hyphenated techniques. Many hyphenated instruments are that have been developed and published for a wide var-
commercially available. These types of instruments for use iety of analytes in many different matrices. These methods
in the pharmaceutical industry have been designed to pro- may be found in the chemical literature; in journals such
cess samples in very large batches in a completely automated as Analytical Chemistry, The Analyst, Analytical and
fashion. The instruments will analyze the samples, store Bioanalytical Chemistry (formerly Fresenius’ Journal of
the data in computer files, “pattern-match” the spectra to Analytical Chemistry), and Talanta; and in journals that
identify the compounds, and calculate the concentrations of focus on specific analytical techniques, such as Applied
the compounds in the samples. Even then, more than one Spectroscopy, Journal of Separation Science (formerly
instrument is required to keep up with the need for charac- Journal of High Resolution Chromatography), Journal
terization of potential drug candidates. As an example, one of the American Society for Mass Spectrometry, and
research department in a major pharmaceutical company Thermochimica Acta. Compilations of “standard” methods
bought its first LC-MS in 1989. By 1998, that one depart- or “official” methods have been published by govern-
ment had more than 40 LC-MS instruments running on a ment agencies, such as the US Environmental Protection
daily basis to support drug metabolism studies alone. Agency (EPA), and private standards organizations, such as
Instrumental methods differ in their ability to do quanti- the American Association of Official Analytical Chemists
tative analysis; some methods are more sensitive than others. (AOAC), the American Society for Testing and Materials
That is, some methods can detect smaller amounts of a given (ASTM), and the American Public Health Association
analyte than other methods. Some methods are useful for (APHA), among others. Similar organizations and offi-
wide ranges of analyte concentrations; other methods have cial methods exist in many other countries. These standard
very limited ranges. We will discuss the reasons for this in the methods are methods that have been tested by many labora-
chapters on the individual techniques, but Table 1.3 shows tories and have been found to be reproducible, with known
the approximate useful concentration ranges for common accuracy and precision. The bibliography lists several of
instrumental techniques. Table 1.3 is meant to serve as a these books on analytical methods. It is always a good idea
guide; the actual sensitivity and useful concentration range to check the chemical literature first, so that you don’t waste
(also called the working range) of a technique for a specific time designing a procedure that already exists.
analysis will depend on many factors.
Note: Throughout this book we mention the standards
1.2.2 Designing the Analytical Method and procedures of ASTM International. Prior to 2001,
the organization was known as the American Society for
Once the problem has been defined, an analytical pro- Testing and Materials (hence, ASTM). First formed in
cedure, or method, must be designed to solve the problem. 1898 under the leadership of Charles Benjamin Dudley,
The analytical chemist may have to design the method to the organization first addressed railroad component quality.
meet certain goals, such as achieving a specified accuracy Today, it is the primary international standards organiza-
and precision, using only a limited amount of sample, or tion in the United States; similar bodies reside in nearly
performing the analysis within a given cost limit or “turn- all major industrialized countries. ASTM International
around time.” Turnaround time is the time elapsed from presides over the adoption of consensus standards by
receipt of a sample in the lab to delivery of the results to supporting thousands of volunteer technical committees,
the person who requested the analysis. This length of time with international membership. Typical committee make
may need to be very short for clinical chemistry labora- up includes members from industry (suppliers, service
tories providing support to hospital emergency rooms, for providers, original equipment manufacturers, users),
example. A common goal for modern analytical procedures members from government laboratories, and members from
universities. ASTM International technical committees and established, unknown samples can be measured and the
subcommittees maintain approximately 12,000 standard analyte concentrations determined. Analytical methods
procedures and practices, published periodically in the should require some sort of reference standard or check
82 volume “Annual Book of Standards.” The deliberative standard. This is also a standard of known composition with
nature and ballot process of ASTM International is inten- a known concentration of the analyte. This check standard
tionally long and careful, sometimes giving rise to criticism is not one of the calibration standards and should be from
of the organization as reactive rather than proactive. a different lot of material than the calibration standards. It
is run as a sample to confirm that the calibration is correct
If there are no methods available, then the analytical and to assess the accuracy and precision of the analysis.
chemist must develop a method to perform the analysis. Reference standard materials are available from govern-
For very challenging problems, this may mean inventing ment and private sources in many countries. Examples of
entirely new analytical instruments or modifying existing government sources are the NIST in the United States, the
instruments to handle the task. National Research Council of Canada (NRCC), and the
The design of the method also requires the analyst to LGC (formerly Laboratory of the Government Chemist) in
consider how the method will be shown to be accurate and the United Kingdom.
precise. This requires knowledge of how we assess accuracy
and precision, discussed in Section 1.3. The analyst must 1.2.3 Sampling
evaluate interferences. Interference is anything that (1) gives
a response other than the analyte itself or (2) changes One of the most important single steps in an analysis is
the response of the analyte. Interferences may be other collecting the sample of the material to be analyzed. Often,
compounds or elements present in the sample or that form on this step is carried out not by the personnel performing the
degradation of the sample. Interfering compounds or elem- analyses, but rather by field technicians. Real materials are
ents may respond directly in the instrumental measurement usually not homogeneous, so the sample must be chosen
to give a false analyte signal, or they may affect the response carefully to be representative of the real material. A repre-
of the analyte indirectly by enhancing or suppressing the sentative sample is one that reflects the true value and distri-
analyte signal. Examples will be given in the chapters for bution of the analyte in the original material. If the sample
each instrumental technique. The analyst must demonstrate is not taken properly, no matter how excellent the analyt-
that the method is reliable and robust. These will be covered ical method or how expert the analyst, the result obtained
in greater detail in Sections 1.5 and 1.6. will not provide a reliable characterization of the material.
There are some fundamental features that should be part Other scientists, law enforcement officials, and medical
of every good analytical method. The method should require professionals often collect samples for analysis, sometimes
that a blank be prepared and analyzed. A blank is used to with no training in how to take a proper sample. The analyt-
ascertain and correct for certain interferences in the ana- ical chemist ideally would be part of the team that discusses
lysis. In many cases, more than one type of blank is needed. collection of samples before they are taken, but in reality,
One type of blank solution may be just the pure solvent used samples often “show up” in the lab. It is important that the
for the sample solutions. This will ensure that no analyte is analyst talks with the sample collector before doing any
present in the solvent and allows the analyst to set the base- analyses; if the sample has been contaminated or improp-
line or the “zero point” in many analyses. A reagent blank erly stored, the analysis will be not only a waste of time but
may be needed; this blank contains all of the reagents used can also lead to erroneous conclusions. In clinical chemistry
to prepare the sample but does not contain the sample itself. analysis, this could lead to a misdiagnosis of a disease; in
Again, this assures the analyst that none of the reagents forensic analysis, this could lead to a serious miscarriage
themselves contribute analyte to the final reported value of of justice.
analyte in the sample. Sometimes a matrix blank is needed; The amount of sample taken must be sufficient for all
this is a blank that is similar in chemical composition to the analyses to be carried out in duplicate or triplicate, if pos-
sample but without the analyte. It may be necessary to use sible. Of course, if only a small quantity of sample is avail-
such a blank to correct for an overlapping spectral line from able, as may be the case for forensic samples from a crime
the matrix in AES, for example. scene or rocks brought back from the moon, the analyst
All instrumental analytical methods except coulometry must do the best job possible with what is provided.
(Chapter 15) require calibration standards, which have A good example of the problems encountered in sam-
known concentrations of the analyte present in them. (In pling real materials is collecting a sample of a metal or metal
isotope dilution MS [IDMS], there must be a known amount alloy. When a molten metal solidifies, the first portion of
of an isotope, usually of the analyte.) These calibration solid to form tends to be the most pure (remember freezing
standards are used to establish the relationship between point depression from your general chemistry class?). The
the analytical signal being measured by the instrument and last portion to solidify is the most impure and is generally
the concentration of the analyte. Once this relationship is located in the center or core of the solidified metal. It is
important to bear this in mind when sampling solid metals. discarded. This process is shown in Figure 1.3. This process
A sample is often ground from a representative cross section can be repeated until a sample of a suitable size for analysis
of the solid, or a hole is drilled through a suitable location is obtained. This sample can still be very large. Ferroalloys,
and the drillings mixed and used as the sample. for example, are highly segregated (i.e., inhomogeneous)
Samples have to be collected using some type of materials; it is not uncommon for the amount required for a
collection tool and put into some type of container. These representative sample of alloy in pieces about 2 in. in diam-
tools and containers can often contaminate the sample. For eter to be 1 ton (0.9 Mg) of material from the lot of alloy.
example, stainless steel needles can add traces of metals to A computer program that generates random numbers
blood or serum samples. Metal spatulas, scissors, drill bits, can choose the sampling locations and is very useful for
glass pipettes, filter paper, and plastic and rubber tubing environmental and agricultural sampling. If the lot is a field
can add unwanted inorganic and organic contaminants to of corn, for example, the field can be divided into a grid, with
samples. To avoid iron, nickel, and chromium contamin- each grid division given a number. The computer program
ation from steel, some implements like tongs and tweezers can pick the random grid divisions to be sampled. Then a
can be purchased with platinum or gold tips. smaller, homogeneous laboratory sample is prepared from
The discussion of sampling, which follows, refers to the the gross composite sample. If the sample is segregated (i.e.,
traditional process of collecting a sample at one location highly inhomogeneous), the representative sample must be
(often called “collection in the field”) and transporting the a composite sample that reflects each region and its relative
sample to the laboratory at a different location. Today, it amount. This is often not known, resulting in the require-
is often possible to analyze samples in situ or during the ment for very large samples. The smaller laboratory sample
production of the material (online or process analysis) may be obtained by several methods but must be repre-
with suitable instrumental probes, completely eliminating sentative of the lot and large enough to provide sufficient
the need for “collecting” a sample. Examples of in situ material for all the necessary analyses. After the labora-
and online analysis and field portable instruments will be tory sample is selected, it is usually split into even smaller
discussed in later chapters. test portions. Multiple small test portions of the laboratory
The process of sampling requires several steps, espe- sample are often taken for replicate analyses and for ana-
cially when sampling bulk materials such as coal, metal lysis by more than one technique. The term aliquot is used
ore, soil, grain, and tank cars of oil or chemicals. First, a to refer to a quantitative amount of a dissolved test portion;
gross representative sample is gathered from the lot. The for example, a 0.100 g test portion of sodium chloride may
lot is the total amount of material available. Portions of the be dissolved in water in a volumetric flask to form 100.0 mL
gross sample should be taken from various locations within of test solution. Three 10.0 mL aliquots may be taken with a
the lot to ensure that the gross sample is representative. volumetric pipette for triplicate analysis for chloride using
For very large lots of solid material such as coal or ore, an ion-selective electrode, for example.
the long pile and alternate shovel method can be used. The As the total amount of the sample is reduced, it
material is formed into a long rectangular pile. It is then should be broken down to successively smaller pieces by
separated into two piles by shoveling material first to one grinding, milling, chopping, or cutting. The 1 ton sample
side and then to the other, creating two piles. One pile is of ferroalloy, for example, must be crushed, ground, and
set aside. The remaining pile may be reduced in size by sieved many times. During the process, the sample size is
repeating the process, until a sample of a size to be sent reduced using a sample splitter called a riffle. After all this
to the laboratory remains. The cone and quarter method is and then a final drying step, a 1 lb (454 g) sample remains.
also used to collect a gross sample of solid materials. The The sample must be mixed well during this entire process
sample is made into a circular pile and mixed well. It is to ensure that it remains representative of the original.
then separated into quadrants. A second pile is made up of The grinding equipment used must not contaminate the
two opposite quadrants, and the remainder of the first pile sample. For example, boron carbide and tungsten carbide
are often used in grinding samples because they are very
hard materials, harder than most samples. However, they
Discard opposite can contribute boron or tungsten to the ground sample, so
quarters
would not be used if boron or tungsten must be measured
at low concentrations. Zirconium oxide ball mills can con-
tribute Zr and Hf to a sample. Stainless steel grinders are a
source of Fe, Cr, and Ni. Some cutting devices use organic
Side view Smaller cone
fluids as lubricants; these must be removed from the sample
Top view before analysis.
It is also possible for the grinding or milling step to cause
erroneously low results for some analytes. Malleable metals
Figure 1.3 The cone and quarter method of sampling bulk
materials. like gold may adhere to the grinding or milling surface and
Size I Size II Size III

(accurate representation (accurate representation (may be difficult to mix properly)
requires too large a sample) can be achieved with a
reasonably sized sample)
Figure 1.4 Sampling of a segregated material with a problematic component like gold. (Extracted from Dulski, T.R., A Manual for the
Chemical Analysis of Metals, ASTM International, West Conshohocken, PA, 1996. With permission. Copyright 1996 ASTM
International.)
be removed from the sample in the process, an undesirable Standard sampling procedures for many materials can be
effect. An example of sampling a segregated material with a found in the Annual Book of ASTM Standards, for example.
problematic component like gold is illustrated in Figure 1.4. Sampling procedures for soil, water, and air are established
The rectangular piece at the top is a hypothetical piece of by the US EPA in the United States and similar government
gold-bearing quartz. The gold is represented as the dark organizations in other countries. Procedures for sampling of
flecks. You can see that the gold appears in bands within water and wastewater can be found in Standards Methods
the quartz, separated by bands of pure quartz (the white for the Analysis of Water and Wastewater; the AOAC
area). If the rock is crushed to Size I, the gold particles have publishes procedures for food products. The bibliography
not been liberated from the quartz; some pieces have gold provides some examples of these publications. A good ana-
flecks and many large pieces are pure quartz. At this size, it lytical chemist will consult the literature before sampling an
is difficult to remove a sample of the rock pieces and expect unfamiliar material. Some general guidelines for sampling
it to be representative. If the rock is crushed to a smaller different classes of materials are discussed here.
size, Size II, it is evident that a representative small sample
can be obtained. If the rock is crushed to Size III, the gold 1.2.3.1 Gas Samples
particles are freed from the quartz matrix. If this sample
could be mixed perfectly, a smaller sample could be taken to Gas samples are generally considered homogeneous,
represent the whole than the sample needed at Size II. (Why but gas mixtures may separate into layers of differing
would this be desirable? The smaller the analytical sample, density. Samples that have been collected and allowed to
the less gold is used up by analysis. This is an important settle will need to be stirred before a portion is taken for
consideration with valuable analytes and valuable samples.) analysis. Gas samples can be taken at a single point in time
But the gold particles and the quartz particles have different (called a grab sample) or can be collected over a period of
densities and shapes and will be difficult to mix well. As time or from different locations to provide an average or
mentioned earlier, gold is soft and malleable. If it is broken composite sample. Gas samples can be collected using gas-
out of the quartz, it may become embedded in the grinder or tight syringes, balloons, plastic bags, or containers made
smeared onto surfaces in the grinding equipment, so some of metal or glass that can be evacuated. Sampling of toxic,
gold may actually be lost from the sample particles ground flammable, or corrosive gases should be done with great
to Size III. Size II will give a more representative sample care using appropriate safety equipment.
than either of the other sizes. The containers used to collect the samples must not con-
Sampling procedures for industrial materials, environ- taminate the sample with analyte. Plastic bags and balloons
mental samples, and biological samples are often agreed may leach volatile organic compounds into the gas sample,
upon, or standardized, by industry, government, and pro- while glass may adsorb components of the sample onto the
fessional societies. Standard sampling procedures help to surface of the glass.
ensure that the samples analyzed are representative and are Certain components of gas samples, such as organic
not contaminated or changed during the sampling process. vapors in air, may be collected by pulling the air through
activated charcoal. The organic gases are adsorbed of oil and water from an oil spill at sea, oil and vinegar
onto the charcoal, while the majority of the air (oxygen, salad dressing, or cream at the top of a bottle of milk. The
nitrogen, etc.) passes through. This has the advantage of layers may need to be emulsified to provide a representa-
preconcentrating the analytes of interest and reducing the tive sample, but it may be more useful to sample each layer
physical size of the sample. Many liters of air can be pulled separately.
through an activated charcoal bed that is no bigger than a Sampling of hot molten materials such as metals, alloys,
ball-point pen. It is much easier to transport the analytes and glasses is a form of liquid sampling, but one requiring
trapped on the charcoal to the laboratory than to transport very specialized equipment and techniques.
hundreds of liters of air. The process of trapping an analyte
out of the gas phase is called “scrubbing.” Scrubbing a gas 1.2.3.3 Solid Samples
sample can also be done by bubbling the gas through a
liquid that will absorb the analytes of interest. Solid samples are often the most difficult to sample
Gas samples may contain particles of solid material because they are usually less homogeneous than gases
that need to be removed by filtration. The filter material or liquids. Large amounts of solid sample cannot be con-
must be chosen so that it does not adsorb analytes or add veniently “stirred up.” Moreover, unlike the situation with
contaminants to the gas. Filters are available that will fluids, there are no diffusion or convection currents in solids
remove particles as small as 0.2 μm in diameter from a gas to ensure mixing. Solids must often be ground or drilled or
stream. crushed into smaller particles to homogenize the sample.
There are many types of manual and automated grinders and
1.2.3.2 Liquid Samples crushers available; the choice depends on the hardness of the
material to be ground. Soft materials also pose a challenge
Liquid samples can also be collected as grab samples or in grinding because they often just deform instead of being
as composite samples. Sampling liquids can be quite diffi- reduced in size. Polymer pellets may be ground in an elec-
cult; it is not always as straightforward as “pouring some” tric coffee grinder with a small amount of liquid nitrogen
out of a bottle or dipping a bucket into a fluid. Only a few added to the grinder (called cryogrinding). The liquid
comments with respect to general sampling of liquids can nitrogen freezes the polymer, making the pellets brittle and
be made here. It is usual to stir liquid samples adequately capable of being easily powdered. Other soft solids such as
to obtain a representative sample; however, there may be foods can be handled the same way. Commercial cryomills
occasions when stirring is not desired. If the analyst is only that prevent the user from coming into contact with liquid
interested in identifying an oily layer floating on water, nitrogen are available. Many solid materials must be oven-
stirring the sample is not needed; the oily layer may be dried before sampling to remove adsorbed water in order
pulled off with a pipette or an eyedropper, for example. to obtain a representative sample. There are numerous
Samples must be collected at locations remote from sources published standard methods for sampling solid materials
of contamination if a representative sample is desired. For such as cement, textiles, food, soil, ceramics, and other
example, if a sample of “normal” river water is desired, materials. Examples of the wide variety of analytical pul-
the sample should be collected away from riverbanks, verizing, grinding, and blending equipment available can be
floating froth, oil, and discharges from industrial and muni- found at the following websites: SpexCertiprep (www.spex
cipal waste treatment sites. Sampling of rivers, lakes, and csp.com), Retsch (www.retsch.com), and Netzsch (www.
similar bodies of water may require samples from different netzsch.com), among others.
depths or different distances from shore. Such samples may
be analyzed individually or blended to obtain an average 1.2.4 Storage of Samples
composition.
Liquid samples may contain particles of solid material When samples cannot be analyzed immediately, they
that need to be removed by filtration or centrifugation. The must be stored. The composition of a sample may change
filter material must be chosen so that it does not adsorb during storage because of reactions with air, light, or inter-
analytes or contaminate the liquid. Some samples that are action with the container material. The container used
mostly liquid contain suspended solid material; orange juice for collection and storage of the sample and the storage
and liquid antacids are examples. In these types of samples, conditions must be chosen to minimize changes in the
the liquid and its associated solids may need to be sampled sample.
for analysis without removing the solids. It may be difficult Plastic containers may leach organic components such as
to obtain a representative sample from these suspensions; a plasticizers and monomers into a sample. Plastic containers
standard sampling procedure is needed to ensure that results may also introduce trace metal impurities such as Cu, Mn,
can be compared from one day to the next. Liquid samples or Pt from the catalysts used to make the polymer or elem-
may consist of more than one layer because they contain ents such as Si, Ti, Sb, Br, and P from inorganic fillers and
two or more immiscible liquids. Examples include samples flame retardants. Glass surfaces both adsorb and release
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6. Mrs. Greene’s will makes the five children equal beneficiaries.
In event of death of any of them the survivors share alike; and if all
should die the estate goes to their families, if any.
7. The sleeping-rooms of the Greenes are arranged thus: Julia’s
and Rex’s face each other at the front of the house; Chester’s and
Ada’s face each other in the centre of the house; and Sibella’s and
Mrs. Greene’s face each other at the rear. No two rooms
intercommunicate, with the exception of Ada’s and Mrs. Greene’s;
and these two rooms also give on the same balcony.
8. The library of Tobias Greene, which Mrs. Greene believes she
had kept locked for twelve years, contains a remarkably complete
collection of books on criminology and allied subjects.
9. Tobias Greene’s past was somewhat mysterious, and there
were many rumors concerning shady transactions carried on by him
in foreign lands.
First Crime
10. Julia is killed by a contact shot, fired from the front, at 11.30 P.
M.
11. Ada is shot from behind, also by a contact shot. She
recovers.
12. Julia is found in bed, with a look of horror and amazement on
her face.
13. Ada is found on the floor before the dressing-table.
14. The lights have been turned on in both rooms.
15. Over three minutes elapse between the two shots.
16. Von Blon, summoned immediately, arrives within half an hour.
17. A set of footprints, other than Von Blon’s, leaving and
approaching the house, is found; but the character of the snow
renders them indecipherable.
18. The tracks have been made during the half-hour preceding
the crime.
19. Both shootings are done with a .32 revolver.
20. Chester reports that an old .32 revolver of his is missing.
21. Chester is not satisfied with the police theory of a burglar, and
insists that the District Attorney’s office investigate the case.
22. Mrs. Greene is aroused by the shot fired in Ada’s room, and
hears Ada fall. But she hears no footsteps or sound of a door
closing.
23. Sproot is half-way down the servants’ stairs when the second
shot is fired, yet he encounters no one in the hall. Nor does he hear
any noise.
24. Rex, in the room next to Ada’s, says he heard no shot.
25. Rex intimates that Chester knows more about the tragedy
than he admits.
26. There is some secret between Chester and Sibella.
27. Sibella, like Chester, repudiates the burglar theory, but
refuses to suggest an alternative, and says frankly that any member
of the Greene family may be guilty.
28. Ada says she was awakened by a menacing presence in her
room, which was in darkness; that she attempted to run from the
intruder, but was pursued by shuffling footsteps.
29. Ada says a hand touched her when she first arose from bed,
but refuses to make any attempt to identify the hand.
30. Sibella challenges Ada to say that it was she (Sibella) who
was in the room, and then deliberately accuses Ada of having shot
Julia. She also accuses Ada of having stolen the revolver from
Chester’s room.
31. Von Blon, by his attitude and manner, reveals a curious
intimacy between Sibella and himself.
32. Ada is frankly fond of Von Blon.
Second Crime
33. Four days after Julia and Ada are shot, at 11.30 p. m., Chester
is murdered by a contact shot fired from a .32 revolver.
34. There is a look of amazement and horror on his face.
35. Sibella hears the shot and summons Sproot.
36. Sibella says she listened at her door immediately after the
shot was fired, but heard no other sound.
37. The lights are on in Chester’s room. He was apparently
reading when the murderer entered.
38. A clear double set of footprints is found on the front walk. The
tracks have been made within a half-hour of the crime.
39. A pair of galoshes, exactly corresponding to the footprints, is
found in Chester’s clothes-closet.
40. Ada had a premonition of Chester’s death, and, when
informed of it, guesses he has been shot in the same manner as
Julia. But she is greatly relieved when shown the footprint patterns
indicating that the murderer is an outsider.
41. Rex says he heard a noise in the hall and the sound of a door
closing twenty minutes before the shot was fired.
42. Ada, when told of Rex’s story, recalls also having heard a
door close at some time after eleven.
43. It is obvious that Ada knows or suspects something.
44. The cook becomes emotional at the thought of any one
wanting to harm Ada, but says she can understand a person having
a reason to shoot Julia and Chester.
45. Rex, when interviewed, shows clearly that he thinks some
one in the house is guilty.
46. Rex accuses Von Blon of being the murderer.
47. Mrs. Greene makes a request that the investigation be
dropped.
Third Crime
48. Rex is shot in the forehead with a .32 revolver, at 11.20 a. m.,
twenty days after Chester has been killed and within five minutes of
the time Ada phones him from the District Attorney’s office.
49. There is no look of horror or surprise on Rex’s face, as was
the case with Julia and Chester.
50. His body is found on the floor before the mantel.
51. A diagram which Ada asked him to bring with him to the
District Attorney’s office has disappeared.
52. No one up-stairs hears the shot, though the doors are open;
but Sproot, down-stairs in the butler’s pantry, hears it distinctly.
53. Von Blon is visiting Sibella that morning; but she says she
was in the bathroom bathing her dog at the time Rex was shot.
54. Footprints are found in Ada’s room coming from the balcony
door, which is ajar.
55. A single set of footprints is found leading from the front walk
to the balcony.
56. The tracks could have been made at any time after nine
o’clock that morning.
57. Sibella refuses to go away on a visit.
58. The galoshes that made all three sets of footprints are found
in the linen-closet, although they were not there when the house was
searched for the revolver.
59. The galoshes are returned to the linen-closet, but disappear
that night.
Fourth Crime
60. Two days after Rex’s death Ada and Mrs. Greene are
poisoned within twelve hours of each other—Ada with morphine,
Mrs. Greene with strychnine.
61. Ada is treated at once, and recovers.
62. Von Blon is seen leaving the house just before Ada swallows
the poison.
63. Ada is discovered by Sproot as a result of Sibella’s dog
catching his teeth in the bell-cord.
64. The morphine was taken in the bouillon which Ada habitually
drank in the mornings.
65. Ada states that no one visited her in her room after the nurse
had called her to come and drink the bouillon; but that she went to
Julia’s room to get a shawl, leaving the bouillon unguarded for
several moments.
66. Neither Ada nor the nurse remembers having seen Sibella’s
dog in the hall before the poisoned bouillon was taken.
67. Mrs. Greene is found dead of strychnine-poisoning the
morning after Ada swallowed the morphine.
68. The strychnine could have been administered only after 11 p.
m. the previous night.
69. The nurse was in her room on the third floor between 11 and
11.30 p. m.
70. Von Blon was calling on Sibella that night, but Sibella says he
left her at 10.45.
71. The strychnine was administered in a dose of citrocarbonate,
which, presumably, Mrs. Greene would not have taken without
assistance.
72. Sibella decides to visit a girl chum in Atlantic City, and leaves
New York on the afternoon train.
Distributable Facts
73. The same revolver is used on Julia, Ada, Chester, and Rex.
74. All three sets of footprints have obviously been made by
some one in the house for the purpose of casting suspicion on an
outsider.
75. The murderer is some one whom both Julia and Chester
would receive in their rooms, in negligé, late at night.
76. The murderer does not make himself known to Ada, but
enters her room surreptitiously.
77. Nearly three weeks after Chester’s death Ada comes to the
District Attorney’s office, stating she has important news to impart.
78. Ada says that Rex has confessed to her that he heard the
shot in her room and also heard other things, but was afraid to admit
them; and she asks that Rex be questioned.
79. Ada tells of having found a cryptic diagram, marked with
symbols, in the lower hall near the library door.
80. On the day of Rex’s murder Von Blon reports that his
medicine-case has been rifled of three grains of strychnine and six
grains of morphine—presumably at the Greene mansion.
81. The library reveals the fact that some one has been in the
habit of going there and reading by candle-light. The books that
show signs of having been read are: a handbook of the criminal
sciences, two works on toxicology, and two treatises on hysterical
paralysis and sleep-walking.
82. The visitor to the library is some one who understands
German well, for three of the books that have been read are in
German.
83. The galoshes that disappeared from the linen-closet on the
night of Rex’s murder are found in the library.
84. Some one listens at the door while the library is being
inspected.
85. Ada reports that she saw Mrs. Greene walking in the lower
hall the night before.
86. Von Blon asserts that Mrs. Greene’s paralysis is of a nature
that makes movement a physical impossibility.
87. Arrangements are made with Von Blon to have Doctor
Oppenheimer examine Mrs. Greene.
88. Von Blon informs Mrs. Greene of the proposed examination,
which he has scheduled for the following day.
89. Mrs. Greene is poisoned before Doctor Oppenheimer’s
examination can be made.
90. The post mortem reveals conclusively that Mrs. Greene’s leg
muscles were so atrophied that she could not have walked.
91. Ada, when told of the autopsy, insists that she saw her
mother’s shawl about the figure in the hall, and, on being pressed,
admits that Sibella sometimes wore it.
92. During the questioning of Ada regarding the shawl Mrs.
Mannheim suggests that it was she herself whom Ada saw in the
hall.
93. When Julia and Ada were shot there were, or could have
been, present in the house: Chester, Sibella, Rex, Mrs. Greene, Von
Blon, Barton, Hemming, Sproot, and Mrs. Mannheim.
94. When Chester was shot there were, or could have been,
present in the house: Sibella, Rex, Mrs. Greene, Ada, Von Blon,
Barton, Hemming, Sproot, and Mrs. Mannheim.
95. When Rex was shot there were, or could have been, present
in the house: Sibella, Mrs. Greene, Von Blon, Hemming, Sproot, and
Mrs. Mannheim.
96. When Ada was poisoned there were, or could have been,
present in the house: Sibella, Mrs. Greene, Von Blon, Hemming,
Sproot, and Mrs. Mannheim.
97. When Mrs. Greene was poisoned there were, or could have
been, present in the house: Sibella, Von Blon, Ada, Hemming,
Sproot, and Mrs. Mannheim.
When Markham had finished reading the summary, he went
through it a second time. Then he laid it on the table.
“Yes, Vance,” he said, “you’ve covered the main points pretty
thoroughly. But I can’t see any coherence in them. In fact, they seem
only to emphasize the confusion of the case.”
“And yet, Markham, I’m convinced that they only need
rearrangement and interpretation to be perfectly clear. Properly
analyzed, they’ll tell us everything we want to know.”
Markham glanced again through the pages.
“If it wasn’t for certain items, we could make out a case against
several people. But no matter what person in the list we may assume
to be guilty, we are at once confronted by a group of contradictory
and insurmountable facts. This précis could be used effectively to
prove that every one concerned is innocent.”
“Superficially it appears that way,” agreed Vance. “But we first
must find the generating line of the design, and then relate the
subsidi’ry forms of the pattern to it.”
Markham made a hopeless gesture.
“If only life were as simple as your æsthetic theories!”
“It’s dashed simpler,” Vance asserted. “The mere mechanism of a
camera can record life; but only a highly developed creative
intelligence, with a profound philosophic insight, can produce a work
of art.”
“Can you make any sense—æsthetic or otherwise—out of this?”
Markham petulantly tapped the sheets of paper.
“I can see certain traceries, so to speak—certain suggestions of a
pattern; but I’ll admit the main design has thus far eluded me. The
fact is, Markham, I have a feeling that some important factor in this
case—some balancing line of the pattern, perhaps—is still hidden
from us. I don’t say that my résumé is insusceptible of interpretation
in its present state; but our task would be greatly simplified if we
were in possession of the missing integer.”
Fifteen minutes later, when we had returned to Markham’s main
office, Swacker came in and laid a letter on the desk.
“There’s a funny one, Chief,” he said.
Markham took up the letter and read it with a deepening frown.
When he had finished, he handed it to Vance. The letter-head read,
“Rectory, Third Presbyterian Church, Stamford, Connecticut”; the
date was the preceding day; and the signature was that of the
Reverend Anthony Seymour. The contents of the letter, written in a
small, precise hand, were as follows:
The Honorable John F.-X. Markham,

Dear Sir: As far as I am aware, I have never betrayed a
confidence. But there can arise, I believe, unforeseen circumstances
to modify the strictness of one’s adherence to a given promise, and
indeed impose upon one a greater duty than that of keeping silent.
I have read in the papers of the wicked and abominable things
that have happened at the Greene residence in New York; and I
have therefore come to the conclusion, after much heart-searching
and prayer, that it is my bounden duty to put you in possession of a
fact which, as the result of a promise, I have kept to myself for over a
year. I would not now betray this trust did I not believe that some
good might possibly come of it, and that you, my dear sir, would also
treat the matter in the most sacred confidence. It may not help you—
indeed, I do not see how it can possibly lead to a solution of the
terrible curse that has fallen upon the Greene family—but since the
fact is connected intimately with one of the members of that family, I
will feel better when I have communicated it to you.
On the night of August 29, of last year, a machine drove up to my
door, and a man and a woman asked that I secretly marry them. I
may say that I am frequently receiving such requests from runaway
couples. This particular couple appeared to be well-bred dependable
people, and I concurred with their wishes, giving them my
assurances that the ceremony would, as they desired, be kept
confidential.
The names that appeared on the license—which had been
secured in New Haven late that afternoon—were Sibella Greene, of
New York City, and Arthur Von Blon, also of New York City.
Vance read the letter and handed it back.

“Really, y’ know, I can’t say that I’m astonished——”
Suddenly he broke off, his eyes fixed thoughtfully before him.
Then he rose nervously and paced up and down.
“That tears it!” he exclaimed.
Markham threw him a look of puzzled interrogation.
“What’s the point?”
“Don’t you see?” Vance came quickly to the District Attorney’s
desk. “My word! That’s the one fact that’s missing from my
tabulation.” He then unfolded the last sheet and wrote:
98. Sibella and Von Blon were secretly married a year ago.
“But I don’t see how that helps,” protested Markham.

“Neither do I at this moment,” Vance replied. “But I’m going to
spend this evening in erudite meditation.”
CHAPTER XXIV.
A Mysterious Trip
(Sunday, December 5)
The Boston Symphony Orchestra was scheduled that afternoon

to play a Bach Concerto and Beethoven’s C-Minor Symphony; and
Vance, on leaving the District Attorney’s office, rode direct to
Carnegie Hall. He sat through the concert in a state of relaxed
receptivity, and afterward insisted on walking the two miles back to
his quarters—an almost unheard-of thing for him.
Shortly after dinner Vance bade me good night and, donning his
slippers and house-robe, went into the library. I had considerable
work to do that night, and it was long past midnight when I finished.
On the way to my room I passed the library door, which had been left
slightly ajar, and I saw Vance sitting at his desk—his head in his
hands, the summary lying before him—in an attitude of oblivious
concentration. He was smoking, as was habitual with him during any
sort of mental activity; and the ash-receiver at his elbow was filled
with cigarette-stubs. I moved on quietly, marvelling at the way this
new problem had taken hold of him.
It was half past three in the morning when I suddenly awoke,
conscious of footsteps somewhere in the house. Rising quietly, I
went into the hall, drawn by a vague curiosity mingled with
uneasiness. At the end of the corridor a panel of light fell on the wall,
and as I moved forward in the semidarkness I saw that the light
issued from the partly open library door. At the same time I became
aware that the footsteps, too, came from that room. I could not resist
looking inside; and there I saw Vance walking up and down, his chin
sunk on his breast, his hands crammed into the deep pockets of his
dressing-gown. The room was dense with cigarette-smoke, and his
figure appeared misty in the blue haze. I went back to bed and lay
awake for an hour. When finally I dozed off it was to the
accompaniment of those rhythmic footfalls in the library.
I rose at eight o’clock. It was a dark, dismal Sunday, and I had my
coffee in the living-room by electric light. When I glanced into the
library at nine Vance was still there, sitting at his desk. The reading-
lamp was burning, but the fire on the hearth had died out. Returning
to the living-room, I tried to interest myself in the Sunday
newspapers; but after scanning the accounts of the Greene case I lit
my pipe and drew up my chair before the grate.
It was nearly ten o’clock when Vance appeared at the door. All
night he had been up, wrestling with his self-imposed problem; and
the devitalizing effects of this long, sleepless concentration showed
on him only too plainly. There were shadowed circles round his eyes;
his mouth was drawn; and even his shoulders sagged wearily. But,
despite the shock his appearance gave me, my dominant emotion
was one of avid curiosity. I wanted to know the outcome of his all-
night vigil; and as he came into the room I gave him a look of
questioning expectancy.
When his eyes met mine he nodded slowly.
“I’ve traced the design,” he said, holding out his hands to the
warmth of the fire. “And it’s more horrible than I even imagined.” He
was silent for some minutes. “Telephone Markham for me, will you,
Van? Tell him I must see him at once. Ask him to come to breakfast.
Explain that I’m a bit fagged.”
He went out, and I heard him calling to Currie to prepare his bath.
I had no difficulty in inducing Markham to breakfast with us after I
had explained the situation; and in less than an hour he arrived.
Vance was dressed and shaved, and looked considerably fresher
than when I had first seen him that morning; but he was still pale,
and his eyes were fatigued.
No mention was made of the Greene case during breakfast, but
when we had sought easy chairs in the library, Markham could
withhold his impatience no longer.
“Van intimated over the phone that you had made something out
of the summary.”
“Yes.” Vance spoke dispiritedly. “I’ve fitted all the items together.
And it’s damnable! No wonder the truth escaped us.”
Markham leaned forward, his face tense, unbelieving.
“You know the truth?”
“Yes, I know,” came the quiet answer. “That is, my brain has told
me conclusively who’s at the bottom of this fiendish affair; but even
now—in the daylight—I can’t credit it. Everything in me revolts
against the acceptance of the truth. The fact is, I’m almost afraid to
accept it. . . . Dash it all, I’m getting mellow. Middle-age has crept
upon me.” He attempted to smile, but failed.
Markham waited in silence.
“No, old man,” continued Vance; “I’m not going to tell you now. I
can’t tell you until I’ve looked into one or two matters. You see, the
pattern is plain enough, but the recognizable objects, set in their new
relationships, are grotesque—like the shapes in an awful dream. I
must first touch them and measure them to make sure that they’re
not, after all, mere abortive vagaries.”
“And how long will this verification take?” Markham knew there
was no use to try to force the issue. He realized that Vance was fully
conscious of the seriousness of the situation, and respected his
decision to investigate certain points before revealing his
conclusions.
“Not long, I hope.” Vance went to his desk and wrote something
on a piece of paper, which he handed to Markham. “Here’s a list of
the five books in Tobias’s library that showed signs of having been
read by the nocturnal visitor. I want those books, Markham—
immediately. But I don’t want any one to know about their being
taken away. Therefore, I’m going to ask you to phone Nurse O’Brien
to get Mrs. Greene’s key and secure them when no one is looking.
Tell her to wrap them up and give them to the detective on guard in
the house with instructions to bring them here. You can explain to
her what section of the book-shelves they’re in.”
Markham took the paper and rose without a word. At the door of
the den, however, he paused.
“Do you think it wise for the man to leave the house?”
“It won’t matter,” Vance told him. “Nothing more can happen there
at present.”
Markham went on into the den. In a few minutes he returned.
“The books will be here in half an hour.”
When the detective arrived with the package Vance unwrapped it
and laid the volumes beside his chair.
“Now, Markham, I’m going to do some reading. You won’t mind,
what?” Despite his casual tone, it was evident that an urgent
seriousness underlay his words.
Markham got up immediately; and again I marvelled at the
complete understanding that existed between these two disparate
men.
“I have a number of personal letters to write,” he said, “so I’ll run
along. Currie’s omelet was excellent.—When shall I see you again? I
could drop round at tea-time.”
Vance held out his hand with a look bordering on affection.
“Make it five o’clock. I’ll be through with my perusings by then.
And thanks for your tolerance.” Then he added gravely: “You’ll
understand, after I’ve told you everything, why I wanted to wait a bit.”
When Markham returned that afternoon a little before five Vance
was still reading in the library; but shortly afterward he joined us in
the living-room.
“The picture clarifies,” he said. “The fantastic images are
gradually taking on the aspect of hideous realities. I’ve substantiated
several points, but a few facts still need corroboration.”
“To vindicate your hypothesis?”
“No, not that. The hypothesis is self-proving. There’s no doubt as
to the truth. But—dash it all, Markham!—I refuse to accept it until
every scrap of evidence has been incontestably sustained.”
“Is the evidence of such a nature that I can use it in a court of
law?”
“That is something I refuse even to consider. Criminal
proceedings seem utterly irrelevant in the present case. But I
suppose society must have its pound of flesh, and you—the duly
elected Shylock of God’s great common people—will no doubt wield
the knife. However, I assure you I shall not be present at the
butchery.”
Markham studied him curiously.
“Your words sound rather ominous. But if, as you say, you have
discovered the perpetrator of these crimes, why shouldn’t society
exact punishment?”
“If society were omniscient, Markham, it would have a right to sit
in judgment. But society is ignorant and venomous, devoid of any
trace of insight or understanding. It exalts knavery, and worships
stupidity. It crucifies the intelligent, and puts the diseased in
dungeons. And, withal, it arrogates to itself the right and ability to
analyze the subtle sources of what it calls ‘crime,’ and to condemn to
death all persons whose inborn and irresistible impulses it does not
like. That’s your sweet society, Markham—a pack of wolves watering
at the mouth for victims on whom to vent its organized lust to kill and
flay.”
Markham regarded him with some astonishment and
considerable concern.
“Perhaps you are preparing to let the criminal escape in the
present case,” he said, with the irony of resentment.
“Oh, no,” Vance assured him. “I shall turn your victim over to you.
The Greene murderer is of a particularly vicious type, and should be
rendered impotent. I was merely trying to suggest that the electric
chair—that touchin’ device of your beloved society—is not quite the
correct method of dealing with this culprit.”
“You admit, however, that he is a menace to society.”
“Undoubtedly. And the hideous thing about it is that this
tournament of crime at the Greene mansion will continue unless we
can put a stop to it. That’s why I am being so careful. As the case
now stands, I doubt if you could even make an arrest.”
When tea was over Vance got up and stretched himself.
“By the by, Markham,” he said offhandedly, “have you received
any report on Sibella’s activities?”
“Nothing important. She’s still in Atlantic City, and evidently
intends to stay there for some time. She phoned Sproot yesterday to
send down another trunkful of her clothes.”
“Did she, now? That’s very gratifyin’.” Vance walked to the door
with sudden resolution. “I think I’ll run out to the Greenes’ for a little
while. I sha’n’t be gone over an hour. Wait for me here, Markham—
there’s a good fellow; I don’t want my visit to have an official flavor.
There’s a new Simplicissimus on the table to amuse you till I return.
Con it and thank your own special gods that you have no Thöny or
Gulbranssen in this country to caricature your Gladstonian features.”
As he spoke he beckoned to me, and, before Markham could
question him, we passed out into the hall and down the stairs.
Fifteen minutes later a taxicab set us down before the Greene
mansion.
Sproot opened the door for us, and Vance, with only a curt
greeting, led him into the drawing-room.
“I understand,” he said, “that Miss Sibella phoned you yesterday
from Atlantic City and asked to have a trunk shipped to her.”
Sproot bowed. “Yes, sir. I sent the trunk off last night.”
“What did Miss Sibella say to you over the phone?”
“Very little, sir—the connection was not good. She said merely
that she had no intention of returning to New York for a considerable
time and needed more clothes than she had taken with her.”
“Did she ask how things were going at the house here?”
“Only in the most casual way, sir.”
“Then she didn’t seem apprehensive about what might happen
here while she was away?”
“No, sir. In fact—if I may say so without disloyalty—her tone of
voice was quite indifferent, sir.”
“Judging from her remarks about the trunk, how long would you
say she intends to be away?”
Sproot considered the matter.
“That’s difficult to say, sir. But I would go so far as to venture the
opinion that Miss Sibella intends to remain in Atlantic City for a
month or more.”
Vance nodded with satisfaction.
“And now, Sproot,” he said, “I have a particularly important
question to ask you. When you first went into Miss Ada’s room on
the night she was shot and found her on the floor before the
dressing-table, was the window open? Think! I want a positive
answer. You know the window is just beside the dressing-table and
overlooks the steps leading to the stone balcony. Was it open or
shut?”
Sproot contracted his brows and appeared to be recalling the
scene. Finally he spoke, and there was no doubt in his voice.
“The window was open, sir. I recall it now quite distinctly. After Mr.
Chester and I had lifted Miss Ada to the bed, I closed it at once for
fear she would catch cold.”
“How far open was the window?” asked Vance with eager
impatience.
“Eight or nine inches, sir, I should say. Perhaps a foot.”
“Thank you, Sproot. That will be all. Now please tell the cook I
want to see her.”
Mrs. Mannheim came in a few minutes later, and Vance indicated
a chair near the desk-light. When the woman had seated herself he
stood before her and fixed her with a stern, implacable gaze.
“Frau Mannheim, the time for truth-telling has come. I am here to
ask you a few questions, and unless I receive a straight answer to
them I shall report you to the police. You will, I assure you, receive
no consideration at their hands.”
The woman tightened her lips stubbornly and shifted her eyes,
unable to meet Vance’s penetrating stare.
“You told me once that your husband died in New Orleans
thirteen years ago. Is that correct?”
Vance’s question seemed to relieve her mind, and she answered
readily.
“Yes, yes. Thirteen years ago.”
“What month?”
“In October.”
“Had he been ill long?”
“About a year.”
“What was the nature of his illness?”
Now a look of fright came into her eyes.
“I—don’t know—exactly,” she stammered. “The doctors didn’t let
me see him.”
“He was in a hospital?”
She nodded several times rapidly. “Yes—a hospital.”
“And I believe you told me, Frau Mannheim, that you saw Mr.
Tobias Greene a year before your husband’s death. That would have
been about the time your husband entered the hospital—fourteen
years ago.”
She looked vaguely at Vance, but made no reply.
“And it was exactly fourteen years ago that Mr. Greene adopted
Ada.”
The woman caught her breath sharply. A look of panic contorted
her face.
“So when your husband died,” continued Vance, “you came to Mr.
Greene, knowing he would give you a position.”
He went up to her and touched her filially on the shoulder.
“I have suspected for some time, Frau Mannheim,” he said kindly,
“that Ada is your daughter. It’s true, isn’t it?”
With a convulsive sob the woman hid her face in her apron.
“I gave Mr. Greene my word,” she confessed brokenly, “that I
wouldn’t tell any one—not even Ada—if he let me stay here—to be
near her.”
“You haven’t told any one,” Vance consoled her. “It was not your
fault that I guessed it. But why didn’t Ada recognize you?”
“She had been away—to school—since she was five.”
When Mrs. Mannheim left us a little later Vance had succeeded in
allaying her apprehension and distress. He then sent for Ada.
As she entered the drawing-room the troubled look in her eyes
and the pallor of her cheeks told clearly of the strain she was under.
Her first question voiced the fear uppermost in her mind.
“Have you found out anything, Mr. Vance?” She spoke with an air
of pitiful discouragement. “It’s terrible alone here in this big house—
especially at night. Every sound I hear . . .”
“You mustn’t let your imagination get the better of you, Ada,”
Vance counselled her. Then he added: “We know a lot more now
than we did, and before long, I hope, all your fears will be done away
with. In fact, it’s in regard to what we’ve found out that I’ve come
here to-day. I thought perhaps you could help me again.”
“If only I could! But I’ve thought and thought. . . .”
Vance smiled.
“Let us do the thinking, Ada.—What I wanted to ask you is this:
do you know if Sibella speaks German well?”
The girl appeared surprised.
“Why, yes. And so did Julia and Chester and Rex. Father insisted
on their learning it. And he spoke it too—almost as well as he spoke
English. As for Sibella, I’ve often heard her and Doctor Von talking in
German.”
“But she spoke with an accent, I suppose.”
“A slight accent—she’d never been long in Germany. But she
spoke very well German.”
“That’s what I wanted to be sure of.”
“Then you do know something!” Her voice quavered with
eagerness. “Oh, how long before this awful suspense will be over?
Every night for weeks I’ve been afraid to turn out my lights and go to
sleep.”
“You needn’t be afraid to turn out your lights now,” Vance assured
her. “There won’t be any more attempts on your life, Ada.”
She looked at him for a moment searchingly, and something in
his manner seemed to hearten her. When we took our leave the
color had come back to her cheeks.
Markham was pacing the library restlessly when we arrived
home.
“I’ve checked several more points,” Vance announced. “But I’ve
missed the important one—the one that would explain the
unbelievable hideousness of the thing I’ve unearthed.”
He went directly into the den, and we could hear him telephoning.
Returning a few minutes later, he looked anxiously at his watch.
Then he rang for Currie and ordered his bag packed for a week’s
trip.
“I’m going away, Markham,” he said. “I’m going to travel—they
say it broadens the mind. My train departs in less than an hour; and
I’ll be away a week. Can you bear to be without me for so long?
However, nothing will happen in connection with the Greene case
during my absence. In fact, I’d advise you to shelve it temporarily.”
He would say no more, and in half an hour he was ready to go.
“There’s one thing you can do for me while I’m away,” he told
Markham, as he slipped into his overcoat. “Please have drawn up for
me a complete and detailed weather report from the day preceding
Julia’s death to the day following Rex’s murder.”
He would not let either Markham or me accompany him to the
station, and we were left in ignorance of even the direction in which
his mysterious trip was to take him.
CHAPTER XXV.
The Capture
(Monday, December 13; 4 p. m.)
It was eight days before Vance returned to New York. He arrived

on the afternoon of Monday, December 13, and, after he had had his
tub and changed his clothes, he telephoned Markham to expect him
in half an hour. He then ordered his Hispano-Suiza from the garage;
and by this sign I knew he was under a nervous strain. In fact, he
had spoken scarcely a dozen words to me since his return, and as
he picked his way down-town through the late afternoon traffic he
was gloomy and preoccupied. Once I ventured to ask him if his trip
had been successful, and he had merely nodded. But when we
turned into Centre Street he relented a little, and said:
“There was never any doubt as to the success of my trip, Van. I
knew what I’d find. But I didn’t dare trust my reason; I had to see the
records with my own eyes before I’d capitulate unreservedly to the
conclusion I’d formed.”
Both Markham and Heath were waiting for us in the District
Attorney’s office. It was just four o’clock, and the sun had already
dropped below the New York Life Building which towered above the
old Criminal Courts structure a block to the southwest.
“I took it for granted you had something important to tell me,” said
Markham; “so I asked the Sergeant to come here.”
“Yes, I’ve much to tell.” Vance had thrown himself into a chair,
and was lighting a cigarette. “But first I want to know if anything has
happened in my absence.”
“Nothing. Your prognostication was quite accurate. Things have
been quiet and apparently normal at the Greene mansion.”

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3.6.2.1 Filters...................................................................................................................................... 102

4.5.4.1 Reaction Kinetics.................................................................................................................... 162

5.3.3.1 Attenuated Total Reflectance.................................................................................................. 206

6.3 Chemical Shifts...................................................................................................................................................... 287

7.2 Instrumentation...................................................................................................................................................... 377

8.1.1 Instrumentation for Flame OES............................................................................................................... 422

8.7 Laser-​Induced Breakdown Spectroscopy.............................................................................................................. 473

9.2.8.4 Quantitative Analysis by XRF................................................................................................ 539

10.3 Ion Mobility Spectrometry.................................................................................................................................... 611

11.4.3.2 Spectral (Isobaric) Interferences............................................................................................. 667

13.7.5 Sulfur–​Phosphorus Flame Photometric Detector.................................................................................... 734

14.5.1 Operating Conditions............................................................................................................................... 792

15.3.3.1 Instrumentation for Conductivity Measurements................................................................... 852

17.2 Differential Thermal Analysis...............................................................................................................................926

Concepts of Instrumental Analytical Chemistry

Figure 1.1 Isomers of erythrose.

Table 1.1 Instrumental Methods of Analysis

Method Elemental Molecular Elemental Molecular

Atomic absorption spectrometry No No Yes No

Table 1.2 Principal Applications of Instrumental Methods of Analysis

Nuclear magnetic resonance (NMR) spectroscopy

Table 1.2 (Continued) Principal Applications of Instrumental Methods of Analysis

Table 1.2 (Continued) Principal Applications of Instrumental Methods of Analysis

Liquid chromatography (LC, high-​performance LC [HPLC])

Table 1.3 Analytical Concentration Ranges for Common Instrumental Methods

X-​ray diffraction No No No Yes Yes

Size I Size II Size III

The Honorable John F.-X. Markham,

Vance read the letter and handed it back.

“But I don’t see how that helps,” protested Markham.

The Boston Symphony Orchestra was scheduled that afternoon

It was eight days before Vance returned to New York. He arrived

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8.7 Laser-Induced Breakdown Spectroscopy.............................................................................................................. 473

13.7.5 Sulfur–Phosphorus Flame Photometric Detector.................................................................................... 734

Figure 1.1 Isomers of erythrose.

Table 1.1 Instrumental Methods of Analysis

Table 1.2 Principal Applications of Instrumental Methods of Analysis

Table 1.2 (Continued) Principal Applications of Instrumental Methods of Analysis

Table 1.2 (Continued) Principal Applications of Instrumental Methods of Analysis

Liquid chromatography (LC, high-performance LC [HPLC])

Table 1.3 Analytical Concentration Ranges for Common Instrumental Methods

X-ray diffraction No No No Yes Yes