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