Handbook of Spectroscopy Edited by G. Gauglitz and T. Vo-Dinh WILE WILEY-VCH Gmbh Co KGaA lSBN3-527-29782-0
Handbook of Spectroscopy Edited by G. Gauglitz and T. Vo-Dinh Handbook of Spectroscopy. Edited by Günter Gauglitz and Tuan Vo-Dinh Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-29782-0
Prof Dr. Guenter Gauglitz This book was carefully produced. Never te for Physical and Theoretical theless, editors, authors and publisher do Chemistry not warrant the information contained of Tubingen therein Readers are Auf der Morgenstelle 8 advised to keep in mind that statements, other items may inadvertently be inaccurate. Prof. Dr. Tuan Vo-Dinh Library of Congress Card No. applied for Advanced Biomedical Science A catalogue record for this book is availabl and Technology Group from the British Library. Oak Ridge National Laboratory P O. Box 2008 Bibliographic information published by Oak Ridge, Tennessee 37831-6101 Die Deutsche Bibliothek USA Die Deutsche Bibliothek lists this publication detailed bibliographic data is available in the E 2003 WILEY-VCH Verlag GmbH& Co KGaA Weinheim All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form sion from the publishers. Registered na trademarks. etc used in this book when not specifically marked as such, not to be considered unprotected by law Printed in the Federal Republic of Germany Printed on acid-free paper. Typesetting Hagedorn Kommunikation rinting Strauss Offsetdruck GmbH sBN3-527-29782-0
Prof. Dr. Guenter Gauglitz Institute for Physical and Theoretical Chemistry University of Tübingen Auf der Morgenstelle 8 72976 Tübingen Germany Prof. Dr. Tuan Vo-Dinh Advanced Biomedical Science and Technology Group Oak Ridge National Laboratory P. O. Box 2008 Oak Ridge, Tennessee 37831-6101 USA This book was carefully produced. Nevertheless, editors, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at http://dnb.ddb.de. 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany. Printed on acid-free paper. Typesetting Hagedorn Kommunikation, Viernheim Printing Strauss Offsetdruck GmbH, Mörlenbach Bookbinding J. Schäffer GmbH & Co. KG, Grünstadt ISBN 3-527-29782-0
Contents Volume 1 Preface XXVIII List of contributors Section I Sample Preparation and Sample Pretreatment 1 Introduction 3 Collection and Preparation of Gaseous Samples 4 Introduction 4 Sampling considerations 5 Active vs Passive Sampling 8 1.3.1 Active air Collection methods 8 1.3.1.1 Sorbents 9 1.3.1.2 Bags 11 1.3.1.3 Canisters 11 1.3.14 Bubblers 12 1.3.1.5 Mist Chambers 13 1.3.2 Passive Sampling 13 Extraction and Preparation of Samples 14 Summary 15 mple Collection and Preparation of Liquid and Solids 17 Introduction 17 Collection of a Representative Sample 1 2.2.1 Statistics of Sampling 18 2.2.2 How Many Samples Should be Obtained? 21 2.2.3 Sampling 2.2.3.1 Liquids 22 2.2.3.2 Preparation of Samples for Analysis 24 时沿 H Verlag gmbh &Co KGaA, Weinheim and luan Vo. Dinh lSBN3-527-29782-0
Contents Volume 1 Preface XXVIII List of Contributors Section I Sample Preparation and Sample Pretreatment 1 Introduction 3 1 Collection and Preparation of Gaseous Samples 4 1.1 Introduction 4 1.2 Sampling considerations 5 1.3 Active vs. Passive Sampling 8 1.3.1 Active Air Collection Methods 8 1.3.1.1 Sorbents 9 1.3.1.2 Bags 11 1.3.1.3 Canisters 11 1.3.1.4 Bubblers 12 1.3.1.5 Mist Chambers 13 1.3.1.6 Cryogenic Trapping 13 1.3.2 Passive Sampling 13 1.4 Extraction and Preparation of Samples 14 1.5 Summary 15 2 Sample Collection and Preparation of Liquid and Solids 17 2.1 Introduction 17 2.2 Collection of a Representative Sample 17 2.2.1 Statistics of Sampling 18 2.2.2 How Many Samples Should be Obtained? 21 2.2.3 Sampling 22 2.2.3.1 Liquids 22 2.2.3.2 Solids 23 2.3 Preparation of Samples for Analysis 24 Contents V Handbook of Spectroscopy. Edited by Günter Gauglitz and Tuan Vo-Dinh Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-29782-0
2.3.1 2.3.1.1 Sample Preparation for Inorganic Analysis 25 3.1.2 Decomposition of Organics 28 2.3.2 iquid Samples 29 2.3.2.1 Extraction/Separation and Preconcentration 29 2.3.2.2Chro Section II Methods 1: Optical Spectroscopy 37 3 Basics of Optical Spectroscopy 39 3.2 Infrared Spectroscopy 41 Raman Spectroscopy 43 3.4 UV/VIS Absorption and Luminescence 44 Instrumentation 48 MIR Spectrometers 4. Dispersive Spectrometers 49 4.1.2 Fourier-Transform Spectrometers 50 4.1.2.1 Detectors 53 4.1.2.2 Step-scan Operation 53 4.1.2.3 Combined technic ques 54 NIR Spectrometers 54 4.2.1 FT-NIR Spectrometers 55 4. 2.2 Scanning- Grating Spectrometers 55 4.2.3 Diode Array Spectrometers 56 4.2.4 4.2.5 LED Spectrometers 56 4.2.6 Raman Spectrometers 57 4.3.1 Raman Grating Spectrometer with Single Channel Detector 57 4.3.1.1 Detectors 59 4.3.1.2 Calibration 60 4.3.2 FT-Raman Spectrometers with Near-Infrared Excitation 61 4.3.3 Raman Grating Polychromator with Multichannel Detector 61 4.4 UV/VIS Spectrometers 63 4.4.1 Sources 64 4.4.2 Monochromators Detectors 64 Fluorescence Spectrometers 66 Measurement Techniques 70 Transmission Measurements 71 Reflection Measurements 73 5.2.1 External Reflection 73
2.3.1 Solid Samples 24 2.3.1.1 Sample Preparation for Inorganic Analysis 25 2.3.1.2 Decomposition of Organics 28 2.3.2 Liquid Samples 29 2.3.2.1 Extraction/Separation and Preconcentration 29 2.3.2.2 Chromatographic Separation 31 Section II Methods 1: Optical Spectroscopy 37 3 Basics of Optical Spectroscopy 39 3.1 Absorption of Light 39 3.2 Infrared Spectroscopy 41 3.3 Raman Spectroscopy 43 3.4 UV/VIS Absorption and Luminescence 44 4 Instrumentation 48 4.1 MIR Spectrometers 48 4.1.1 Dispersive Spectrometers 49 4.1.2 Fourier-Transform Spectrometers 50 4.1.2.1 Detectors 53 4.1.2.2 Step-scan Operation 53 4.1.2.3 Combined Techniques 54 4.2 NIR Spectrometers 54 4.2.1 FT-NIR Spectrometers 55 4.2.2 Scanning-Grating Spectrometers 55 4.2.3 Diode Array Spectrometers 56 4.2.4 Filter Spectrometers 56 4.2.5 LED Spectrometers 56 4.2.6 AOTF Spectrometers 56 4.3 Raman Spectrometers 57 4.3.1 Raman Grating Spectrometer with Single Channel Detector 57 4.3.1.1 Detectors 59 4.3.1.2 Calibration 60 4.3.2 FT-Raman Spectrometers with Near-Infrared Excitation 61 4.3.3 Raman Grating Polychromator with Multichannel Detector 61 4.4 UV/VIS Spectrometers 63 4.4.1 Sources 64 4.4.2 Monochromators 64 4.4.3 Detectors 64 4.5 Fluorescence Spectrometers 66 5 Measurement Techniques 70 5.1 Transmission Measurements 71 5.2 Reflection Measurements 73 5.2.1 External Reflection 73 VI Contents
Contents VII Reflection Absorption 75 5.2.3 Attenuated Total Reflection(ATR)75 5.2.4 Reflection at Thin Films 77 5.2.5 Diffuse Reflection 78 Spectroscopy with Polarized Light 81 5.3.2 Circular Dichroism(CD)82 Photoacoustic measu Microscopic Measurements 84 5.5.1 Infrared Microscopes85 onfocal Microscopes 85 5.5.3 Near-field Microscopes 6 Applications 89 Mid-Infrared(MIR)Spectroscopy 89 6.1.1 Sample Preparation and Measurement 89 6.1.1.1 Gases90 6.1.1.2 Solutions and Neat Liquids 91 6.1.1.3 Pellets and mulls 92 6.1.1.4 Neat Solid Samples 94 6.1. 1.5 Reflection-Absorption Sampling Technique 94 6.1.1.6 Sampling with the ATR Technique 95 6.1. 1.7 Thin Samples 96 6.1. 1.8 Diffuse Reflection Sampling Technique 97 6.1.1.9 Sampling by Photoacoustic Detection 97 6.1.1.10 Microsampling 98 6.1.2 Structural Analysis 98 6.1.2.1 The Region from 4000 to 1400 cm-1 102 6.1.2.2 The Region 1400-900 cm 102 6.1.2.3 The Region from 900 to 400 cm-102 6.1.3 Special Applications 103 Near-Infrared Spectroscopy 104 6.2.2 Applications of NIR Spectroscopy 110 o5 6.2.1 Sample Preparation and Measurement Raman Spectroscopy 112 6.3.1 Sample Preparation and Measurements 112 6.3.1.1 Sample Illumination and Light Collection 113 6.3.1.2 Polarization measurements 118 6.3.1.3 Enhanced Raman Scattering 119 6.3.2 Special Applications 120 UV/VIS Spectroscopy 125 6.4.1 Sample Preparation 125 Structural Analysis 129 6.4.3 Special Applications 132 cence Spectroscopy 135
5.2.2 Reflection Absorption 75 5.2.3 Attenuated Total Reflection (ATR) 75 5.2.4 Reflection at Thin Films 77 5.2.5 Diffuse Reflection 78 5.3 Spectroscopy with Polarized Light 81 5.3.1 Optical Rotatory Dispersion 81 5.3.2 Circular Dichroism (CD) 82 5.4 Photoacoustic Measurements 83 5.5 Microscopic Measurements 84 5.5.1 Infrared Microscopes 85 5.5.2 Confocal Microscopes 85 5.5.3 Near-field Microscopes 86 6 Applications 89 6.1 Mid-Infrared (MIR) Spectroscopy 89 6.1.1 Sample Preparation and Measurement 89 6.1.1.1 Gases 90 6.1.1.2 Solutions and Neat Liquids 91 6.1.1.3 Pellets and Mulls 92 6.1.1.4 Neat Solid Samples 94 6.1.1.5 ReflectionAbsorption Sampling Technique 94 6.1.1.6 Sampling with the ATR Technique 95 6.1.1.7 Thin Samples 96 6.1.1.8 Diffuse Reflection Sampling Technique 97 6.1.1.9 Sampling by Photoacoustic Detection 97 6.1.1.10 Microsampling 98 6.1.2 Structural Analysis 98 6.1.2.1 The Region from 4000 to 1400 cm1 102 6.1.2.2 The Region 1400900 cm1 102 6.1.2.3 The Region from 900 to 400 cm1 102 6.1.3 Special Applications 103 6.2 Near-Infrared Spectroscopy 104 6.2.1 Sample Preparation and Measurement 105 6.2.2 Applications of NIR Spectroscopy 110 6.3 Raman Spectroscopy 112 6.3.1 Sample Preparation and Measurements 112 6.3.1.1 Sample Illumination and Light Collection 113 6.3.1.2 Polarization Measurements 118 6.3.1.3 Enhanced Raman Scattering 119 6.3.2 Special Applications 120 6.4 UV/VIS Spectroscopy 125 6.4.1 Sample Preparation 125 6.4.2 Structural Analysis 129 6.4.3 Special Applications 132 6.5 Fluorescence Spectroscopy 135 Contents VII
Sample Preparation and Measurements 6.5.1.1 Fluorescence Quantum Yield and Lifetime 138 6.5.1.2 Fluores 6.5.1.3 Solvent Relaxation 144 6.5.1.4 Polarized fluorescence 148 6.5.2 Section Ill Methods 2: Nuclear Magnetic Resonance Spectroscopy 169 Introduction 171 NMR Spectroscopy 177 7.1 Introduction Solution-state H NMR 179 73 Solid-state NMR 187 7.3.1 Dipolar Interaction 188 7.3.2 Chemical Shift Anisotropy 190 7.3.3 Quadrupolar Interaction 191 7.3.4 Magic Angle Spinning(MAS) NMR 194 7.3.5 Ti and Tip Relaxation 192 73. Dynamics 198 7.4 Imaging 199 75 D NMR: The HNCA Pulse Sequence 204 7.6 Conclusion 207 8 Solution NMR Spectroscopy 209 Introduction 209 ID(One-dimensional) NMR Methods 210 8.2.1 Proton Spin Decoupling Experiments 211 8.2.2 Proton Decoupled Difference Spectroscopy 212 8.2.3 Nuclear Overhauser Effect(NOE) Difference Spectroscopy 212 2.4 Selective Population Transfer(SPT) 213 8.2.5 J-Modulated Spin Echo Experiments 213 8.2.5. 1 INEPT(Insensitive Nucleus Enhancement by Polarization Transfer) 214 8.2.5. 2 DEPT(Distortionless Enhancement Polarization Transfer)215 8.2.6 Off-Resonance Decoupling 216 8.2.7 Relaxation Measurements 217 Two-dimensional NMR Experiments 218 J-Resolved NMR Experiments 219 8.3.2 Homonuclear 2D NMR Spectroscopy 223 8.3. 2.1 COSY, Homonuclear Correlated Spectroscopy 223 8.3.2.2 Homonuclear TOCSY, Total Correlated Spectroscopy 226 8.3.2.3 NOESY, Nuclear Overhauser Enhancement Spectroscopy 228
6.5.1 Sample Preparation and Measurements 138 6.5.1.1 Fluorescence Quantum Yield and Lifetime 138 6.5.1.2 Fluorescence Quencher 139 6.5.1.3 Solvent Relaxation 144 6.5.1.4 Polarized Fluorescence 148 6.5.2 Special Applications 152 Section III Methods 2: Nuclear Magnetic Resonance Spectroscopy 169 Introduction 171 7 An Introduction to Solution, Solid-State, and Imaging NMR Spectroscopy 177 7.1 Introduction 177 7.2 Solution-state 1 H NMR 179 7.3 Solid-state NMR 187 7.3.1 Dipolar Interaction 188 7.3.2 Chemical Shift Anisotropy 190 7.3.3 Quadrupolar Interaction 191 7.3.4 Magic Angle Spinning (MAS) NMR 194 7.3.5 T1 and T1 Relaxation 195 7.3.6 Dynamics 198 7.4 Imaging 199 7.5 3D NMR: The HNCA Pulse Sequence 204 7.6 Conclusion 207 8 Solution NMR Spectroscopy 209 8.1 Introduction 209 8.2 1D (One-dimensional) NMR Methods 210 8.2.1 Proton Spin Decoupling Experiments 211 8.2.2 Proton Decoupled Difference Spectroscopy 212 8.2.3 Nuclear Overhauser Effect (NOE) Difference Spectroscopy 212 8.2.4 Selective Population Transfer (SPT) 213 8.2.5 J-Modulated Spin Echo Experiments 213 8.2.5.1 INEPT (Insensitive Nucleus Enhancement by Polarization Transfer) 214 8.2.5.2 DEPT (Distortionless Enhancement Polarization Transfer) 215 8.2.6 Off-Resonance Decoupling 216 8.2.7 Relaxation Measurements 217 8.3 Two-dimensional NMR Experiments 218 8.3.1 2D J-Resolved NMR Experiments 219 8.3.2 Homonuclear 2D NMR Spectroscopy 223 8.3.2.1 COSY, Homonuclear Correlated Spectroscopy 223 8.3.2.2 Homonuclear TOCSY, Total Correlated Spectroscopy 226 8.3.2.3 NOESY, Nuclear Overhauser Enhancement Spectroscopy 228 VIII Contents
8.3. 2.4 ROESY, Rotating Frame Overhauser Enhanced Spectroscopy 230 8. 3.2.5 NOESY VS ROESY 231 8.3.2.6 Other Homonuclear Autocorrelation Experiments 23 8.3.3 Gradient Homonuclear 2D NMR Experiments 232 8.3.4 Heteronuclear Shift Correlation 234 8.3.5 Direct heteronuclear Chemical Shift Correlation Methods 234 8.3.5.1 HMQC, Heteronuclear Multiple Quantum Coherence 234 HSQC, Heteronuclear Single Quantum Coherence Chemical Shift 8.3.6.1 Multiplicity-edited Heteronuclear Shift Correlation Experiments 237 8.3.6.2 Accordion-optimized Direct Heteronuclear Shift Correlation Experiments 239 Long-range Heteronuclear Chemical Shift Correlation 240 8.3.7.1 HMBC, Heteronuclear Multiple Bond Correlation 242 8.3.7.2 Variants of the Basic HMBC Experiment 243 8.3.7.3 Accordion-optimized Long-range Heteronuclear Shift Correlation Methods. 244 8.3.7. 4 JJ-HMBC 248 8.3.7.5 Relative Sensitivity of Long-range Heteronuclear Shift Correlation Experiments 251 8.3.7.6 Applications of Accordion-optimized Long-range Heteronuclear Shift Correlation Experiments 252 8.3.8 Hyphenated-2D NMR Experiments 252 8.3.9 One-dimensional Analogues of 2D NMR Experiments 255 8.3.10 Gradient 1D NOESY 255 8.3.11 Selective 1D Long- range Heteronuclear Shift Correlation Experiments 257 8.3.12 Small Sample NMR Studies 257 9 Solid- State NMr 269 Solid-state NMR Lineshapes 272 9.2.1 The Orientational Dependence of the NMR Resonance Frequency 272 9.2.2 Single-crystal NMR 273 9.2.3 Powder Spectra 275 9.2.4 One-dimensional ZH NMR 278 Magic-angle Spinnin 9.3.1 CP MAS NMR 281 9.3.2 H Solid- State Nmr 285 Recoupling Methods 287 9.4.1 Heteronuclear Dipolar-coupled Spins: REDOR 287 Homonuclear Dipolar-coupled Spins 290 94.3 The CSA: CODEX 291 Homonuclear Two-dimensional Experiments 292
8.3.2.4 ROESY, Rotating Frame Overhauser Enhanced Spectroscopy 230 8.3.2.5 NOESY vs. ROESY 231 8.3.2.6 Other Homonuclear Autocorrelation Experiments 231 8.3.3 Gradient Homonuclear 2D NMR Experiments 232 8.3.4 Heteronuclear Shift Correlation 234 8.3.5 Direct Heteronuclear Chemical Shift Correlation Methods 234 8.3.5.1 HMQC, Heteronuclear Multiple Quantum Coherence 234 8.3.6 HSQC, Heteronuclear Single Quantum Coherence Chemical Shift Correlation Techniques 236 8.3.6.1 Multiplicity-edited Heteronuclear Shift Correlation Experiments 237 8.3.6.2 Accordion-optimized Direct Heteronuclear Shift Correlation Experiments 239 8.3.7 Long-range Heteronuclear Chemical Shift Correlation 240 8.3.7.1 HMBC, Heteronuclear Multiple Bond Correlation 242 8.3.7.2 Variants of the Basic HMBC Experiment 243 8.3.7.3 Accordion-optimized Long-range Heteronuclear Shift Correlation Methods. 244 8.3.7.4 2 J 3 J-HMBC 248 8.3.7.5 Relative Sensitivity of Long-range Heteronuclear Shift Correlation Experiments 251 8.3.7.6 Applications of Accordion-optimized Long-range Heteronuclear Shift Correlation Experiments 252 8.3.8 Hyphenated-2D NMR Experiments 252 8.3.9 One-dimensional Analogues of 2D NMR Experiments 255 8.3.10 Gradient 1D NOESY 255 8.3.11 Selective 1D Long-range Heteronuclear Shift Correlation Experiments 257 8.3.12 Small Sample NMR Studies 257 8.4 Conclusions 262 9 Solid-State NMR 269 9.1 Introduction 269 9.2 Solid-state NMR Lineshapes 272 9.2.1 The Orientational Dependence of the NMR Resonance Frequency 272 9.2.2 Single-crystal NMR 273 9.2.3 Powder Spectra 275 9.2.4 One-dimensional 2 H NMR 278 9.3 Magic-angle Spinning 280 9.3.1 CP MAS NMR 281 9.3.2 1 H Solid-State NMR 285 9.4 Recoupling Methods 287 9.4.1 Heteronuclear Dipolar-coupled Spins: REDOR 287 9.4.2 Homonuclear Dipolar-coupled Spins 290 9.4.3 The CSA: CODEX 291 9.5 Homonuclear Two-dimensional Experiments 292 Contents IX
9.5.1 Establishing the Backbone Connectivity in an Organic Molecule 293 9.5.2 Dipolar-mediated Double-quantum Spectroscopy 295 9.5.3 High-resolution H Solid-state NMR 298 9.5.4 Anisotropic Isotropic Correlation: The Measurement of CSAs 300 9.5.5 The Investigation of Slow Dynamics: 2D Exchange 303 H-H DQ MAS Spinning-sideband Patterns 305 Heteronuclear Two-dimensional Experiments 30 9.6.1 Heteronuclear Correlation 307 9.6.2 The Quantitative Determination of Heteronuclear Dipolar Couplings 310 9.6.3 Torsional Angles 9.6.4 Oriented Samples 313 lalf-integer Quadrupole Nuclei 315 319 Section IV Methods 3: Mass Spectrometry 327 Introduction: Principles of Mass Spectrometry 329 10.1.1 Application of Mass Spectrometry to Biopolymer Analysis 330 Techniques and Instrumentation of Mass Spectrometry 331 10.2.1 Sample Introduction and lonisation Methods 331 0. 2.1.1 Pre-conditions 331 10.2. 1.2 Gas Phase("Hard")Ionisation Methods 331 .0.2.1.3 "Soft "Ionisation Techniques 332 10.2.2.1 Magnetic Sector Mass Analysers 335 Analysers 10.2.2.3 Time-of-Flight Mass Analysers 338 10.2.2.4 Trapped-Ion Mass Analysers 339 10.2.2.5 Hybrid Instruments 340 10.2.3 Ion Detection and Spectra Acquisition 340 10.2.4 High Resolution Fourier Transform Ion Cyclotron Resonance(ICR) Mass Spectrometry 341 10.2.5 Sample Preparation and Handling in Bioanalytical Applications 344 10.2.5.1 Liquid-Liquid Extraction(LLE)344 10.2.5.2 Solid Phase Extraction(SPE)343 0. 2.5.3 Immunoaffinity Extraction(IAE)34 10.2.5.4 Solid-phase Microextraction 345 10.2.5.5 Supercritical-Fluid Extraction(SFE)346 10.2.6 Coupling of Mass Spectrometry with Microseparation Methods 346 10.2.6.1 Liquid Chromatography-Mass Spectrometry Coupling(LC-MS)347 10.2.6.2 Capillary Electrophoresis (CE)-Mass Spectrometry 348 10.3 Applications of Mass Spectrometry to Biopolymer Analysis 349
9.5.1 Establishing the Backbone Connectivity in an Organic Molecule 293 9.5.2 Dipolar-mediated Double-quantum Spectroscopy 295 9.5.3 High-resolution 1 H Solid-state NMR 298 9.5.4 Anisotropic – Isotropic Correlation: The Measurement of CSAs 300 9.5.5 The Investigation of Slow Dynamics: 2D Exchange 303 9.5.6 1 H1 H DQ MAS Spinning-sideband Patterns 305 9.6 Heteronuclear Two-dimensional Experiments 307 9.6.1 Heteronuclear Correlation 307 9.6.2 The Quantitative Determination of Heteronuclear Dipolar Couplings 310 9.6.3 Torsional Angles 312 9.6.4 Oriented Samples 313 9.7 Half-integer Quadrupole Nuclei 315 9.8 Summary 319 Section IV Methods 3: Mass Spectrometry 327 10 Mass Spectrometry 329 10.1 Introduction: Principles of Mass Spectrometry 329 10.1.1 Application of Mass Spectrometry to Biopolymer Analysis 330 10.2 Techniques and Instrumentation of Mass Spectrometry 331 10.2.1 Sample Introduction and Ionisation Methods 331 10.2.1.1 Pre-conditions 331 10.2.1.2 Gas Phase (“Hard”) Ionisation Methods 331 10.2.1.3 “Soft” Ionisation Techniques 332 10.2.2 Mass Spectrometric Analysers 335 10.2.2.1 Magnetic Sector Mass Analysers 335 10.2.2.2 Quadrupole Mass Analysers 337 10.2.2.3 Time-of-Flight Mass Analysers 338 10.2.2.4 Trapped-Ion Mass Analysers 339 10.2.2.5 Hybrid Instruments 340 10.2.3 Ion Detection and Spectra Acquisition 340 10.2.4 High Resolution Fourier Transform Ion Cyclotron Resonance (ICR) Mass Spectrometry 341 10.2.5 Sample Preparation and Handling in Bioanalytical Applications 344 10.2.5.1 LiquidLiquid Extraction (LLE) 344 10.2.5.2 Solid Phase Extraction (SPE) 345 10.2.5.3 Immunoaffinity Extraction (IAE) 345 10.2.5.4 Solid-phase Microextraction 345 10.2.5.5 Supercritical-Fluid Extraction (SFE) 346 10.2.6 Coupling of Mass Spectrometry with Microseparation Methods 346 10.2.6.1 Liquid Chromatography-Mass Spectrometry Coupling (LC-MS) 347 10.2.6.2 Capillary Electrophoresis (CE)-Mass Spectrometry 348 10.3 Applications of Mass Spectrometry to Biopolymer Analysis 349 X Contents
10.3.1 Introduction 349 10.3.2 Analysis of Peptide and Protein Primary Structures and post-Translational Structure modifications 34 10.3.3 Tertiary Structure Characterisation by Chemical Modification and Mass Spectrometry 353 10.3.4 Characterisation of Non-Covalent Supramolecular Complexes 354 10.3.5 Mass Spectrometric Proteome Analysis 356 Section V Methods 4: Elemental Analysis 363 X-ray Fluorescence Analysis 365 11.1 Introduction 365 11.2 Basic Principles 367 11.2.1 X-ray Wavelength and Energy Scales 367 11.2.2 Interaction of X-rays with Matter 367 11.2.3 Photoelectric Effect 369 11.2.4 Scattering 371 11.2.5 11.2.6 Selection Rules, Characteristic Lines and X-ray Spectra 3 11.2.7 Figures-of-merit for XRF Spectrometers 376 11.2.7.1 Analytical Sensitivity 376 11.2.7.2 Detection and Determination Limits 377 11.3 Instrumentation 380 11.3.1 X-ray Sources 380 11.3.2 X-ray Detectors 384 11.3.3 Wavelength-dispersive XRF 390 11.3.4 Energy-dispersive XRF 393 11.3.5 Radioisotope XRF 397 11.3.6 Total Reflection XRF 398 Microscopic XRF 399 114 Matrix Effects 40 11.4.1 Thin and Thick Samples 401 11.4.2 Primary and Secondary Absorption, Direct and Third Element Enhancement 403 11.5 Data Treatment 404 11.5.1 Counting Statistics 404 11.5.2 Spectrum Evaluation Techniqu 11.5.2.1 Data Extraction in WDXRF 406 11.5.2.2 Data Extraction in EDXRF: Simple Case, No Peak Overlap 407 11.5.2.3 Data Extraction in EDXRF, Multiple Peak Overlap 408 11.5.3 Quantitative Calibration Procedures 409 11.5.3.1 Single-element Techniques 412 11.5.3. 2 Multiple-element Techniques 413 11.5.4 Error Sources in X-ray Fluorescence Analysis 415
10.3.1 Introduction 349 10.3.2 Analysis of Peptide and Protein Primary Structures and Post-Translational Structure Modifications 349 10.3.3 Tertiary Structure Characterisation by Chemical Modification and Mass Spectrometry 353 10.3.4 Characterisation of Non-Covalent Supramolecular Complexes 354 10.3.5 Mass Spectrometric Proteome Analysis 356 Section V Methods 4: Elemental Analysis 363 11 X-ray Fluorescence Analysis 365 11.1 Introduction 365 11.2 Basic Principles 367 11.2.1 X-ray Wavelength and Energy Scales 367 11.2.2 Interaction of X-rays with Matter 367 11.2.3 Photoelectric Effect 369 11.2.4 Scattering 371 11.2.5 Bremsstrahlung 372 11.2.6 Selection Rules, Characteristic Lines and X-ray Spectra 373 11.2.7 Figures-of-merit for XRF Spectrometers 376 11.2.7.1 Analytical Sensitivity 376 11.2.7.2 Detection and Determination Limits 377 11.3 Instrumentation 380 11.3.1 X-ray Sources 380 11.3.2 X-ray Detectors 384 11.3.3 Wavelength-dispersive XRF 390 11.3.4 Energy-dispersive XRF 393 11.3.5 Radioisotope XRF 397 11.3.6 Total Reflection XRF 398 11.3.7 Microscopic XRF 399 11.4 Matrix Effects 401 11.4.1 Thin and Thick Samples 401 11.4.2 Primary and Secondary Absorption, Direct and Third Element Enhancement 403 11.5 Data Treatment 404 11.5.1 Counting Statistics 404 11.5.2 Spectrum Evaluation Techniques 405 11.5.2.1 Data Extraction in WDXRF 406 11.5.2.2 Data Extraction in EDXRF: Simple Case, No Peak Overlap 407 11.5.2.3 Data Extraction in EDXRF, Multiple Peak Overlap 408 11.5.3 Quantitative Calibration Procedures 409 11.5.3.1 Single-element Techniques 412 11.5.3.2 Multiple-element Techniques 413 11.5.4 Error Sources in X-ray Fluorescence Analysis 415 Contents XI
11.5.5 Specimen Preparation for X-ray Fluorescence 416 11.6 Advantages and Limitations 417 11.6.1 Qualitative Analysis 417 11.6.2 Detection limits 418 11.6.3 Quantitative Reliability 418 117 Atomic Absorption Spectrometry(AAS)and Atomic Emission Spectrometry(AES) 421 12.1 Introduction 421 12.2 Theory of Atomic Spectroscopy 421 12.2.1 Basic Principles 421 12.2.2 Fundamentals of Absorption and Emission 426 12.2. 2.1 Absorption 429 12.2.2.2 Line Broadening 430 12.2. 2.3 Self-absorption 431 12.2.2. 4 Ionisation 432 12.2.2.5 Dissociation 434 12.2.2.6 Radiation Sources and Atom Reservoirs 434 tomic Absorption Spectrometry (AAS)436 12.3.1 Introduction 436 12.3.2 Instrumentation 436 123. 2.1 Radiation Sources 437 12.3. 2.2 Atomisers 440 12.3.2.3 Optical Set-up and Components of Atomic Absorption Instruments 453 12.3.3 Spectral Interference 454 12.3.3.1 Origin of Spectral Interference 454 12.3.3. 2 Methods for Correcting for Spectral Interference 455 123.4 Chemical Interferences 462 12.3.4.1 The Formation of Compounds of Low Volatility 463 12. 3.4.2 Influence on Dissociation Equilibria 463 12.3.4.3 Ionisation in Flames 464 12.3.5 Data Treatment 465 12.3.5.1 Quantitative Analysis 465 12.3.6 Hyphenated Techniques 466 12.3.6.1 Gas Chromatography-Atomic Absorption Spectrometry 467 12.3.6.2 Liquid Chromatography-Atomic Absorption Spectrometry 469 12.3.7 Conclusion and Future Directions 470 tomic Emission Spectrometry(AES)471 12.4.1 Introduction 471 124.2 12. 4.2.1 Atomisation Devices 471 12.4. 2.2 Optical Set-up and Detection 480 12. 4.2.3 Instrumentation for Solid Sample Introduction 483
11.5.5 Specimen Preparation for X-ray Fluorescence 416 11.6 Advantages and Limitations 417 11.6.1 Qualitative Analysis 417 11.6.2 Detection Limits 418 11.6.3 Quantitative Reliability 418 11.7 Summary 419 12 Atomic Absorption Spectrometry (AAS) and Atomic Emission Spectrometry (AES) 421 12.1 Introduction 421 12.2 Theory of Atomic Spectroscopy 421 12.2.1 Basic Principles 421 12.2.2 Fundamentals of Absorption and Emission 426 12.2.2.1 Absorption 429 12.2.2.2 Line Broadening 430 12.2.2.3 Self-absorption 431 12.2.2.4 Ionisation 432 12.2.2.5 Dissociation 434 12.2.2.6 Radiation Sources and Atom Reservoirs 434 12.3 Atomic Absorption Spectrometry (AAS) 436 12.3.1 Introduction 436 12.3.2 Instrumentation 436 12.3.2.1 Radiation Sources 437 12.3.2.2 Atomisers 440 12.3.2.3 Optical Set-up and Components of Atomic Absorption Instruments 453 12.3.3 Spectral Interference 454 12.3.3.1 Origin of Spectral Interference 454 12.3.3.2 Methods for Correcting for Spectral Interference 455 12.3.4 Chemical Interferences 462 12.3.4.1 The Formation of Compounds of Low Volatility 463 12.3.4.2 Influence on Dissociation Equilibria 463 12.3.4.3 Ionisation in Flames 464 12.3.5 Data Treatment 465 12.3.5.1 Quantitative Analysis 465 12.3.6 Hyphenated Techniques 466 12.3.6.1 Gas Chromatography-Atomic Absorption Spectrometry 467 12.3.6.2 Liquid Chromatography-Atomic Absorption Spectrometry 469 12.3.7 Conclusion and Future Directions 470 12.4 Atomic Emission Spectrometry (AES) 471 12.4.1 Introduction 471 12.4.2 Instrumentation 471 12.4.2.1 Atomisation Devices 471 12.4.2.2 Optical Set-up and Detection 480 12.4.2.3 Instrumentation for Solid Sample Introduction 483 XII Contents