Contents

Preface...... xix

Acknowledgments ...... xxiii

Chapter 1 Introduction...... 1 I. Introduction ...... 3 1. The Tools and Data of Mass Spectrometry...... 4 2. The Concept of Mass Spectrometry...... 4 II. History ...... 9 III. Some Important Terminology Used In Mass Spectrometry...... 22 1. Introduction...... 22 2. Ions...... 22 3. Peaks ...... 23 4. Resolution and Resolving Power...... 25 IV. Applications...... 28 1. Example 1-1: Interpretation of Fragmentation Patterns (Mass Spectra) to Distinguish Positional Isomers ...... 29 2. Example 1-2: Drug Overdose: Use of GC/MS to Identify a Drug Metabolite ...... 31 3. Example 1-3: Verification that the Proper Derivative of the Compound of Interest Has Been Prepared...... 32 4. Example 1-4: Use of a CI Mass Spectrum to Complement an EI Mass Spectrum...... 35 5. Example 1-5: Use of Exact Mass Measurements to Identify Analytes According to Elemental Composition...... 38 6. Example 1-6: Is This Protein Phosphorylated? If So, Where? ...... 40 7. Example 1-7: Clinical Diagnostic Tests Based on Quantitation of Stable Isotopes by Mass Spectrometry in Lieu of Radioactivity...... 42 V. The Need for Chromatography ...... 43 VI. Closing Remarks ...... 44 VII. Monographs on Mass Spectrometry Published Before 1970 ...... 45 Chapter 2 TheCOPYRIGHTEDMass Spectrometer ...... MATERIAL...... 53 I. Introduction ...... 55 II. Ion Guides...... 56 III. Types of m/z Analyzers...... 61 1. Time-of-Flight m/z Analyzers...... 62 A. Linear...... 64 1) Resolving Power of the Linear TOF Instrument ...... 65 2) Time-Lag Focusing ...... 66 3) Beam Deflection...... 67 B. Reflectron ...... 69 C. Orthogonal Acceleration ...... 74

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D. Ion Detection in the TOF Analyzer...... 75 1) Time-Slice Detection ...... 76 2) Time-Array Detection ...... 77 3) TAD with Transient Recorders ...... 79 4) TAD with an Integrating Transient Recorder...... 79 5) Hadamard Transform TOF–MS ...... 80 2. Quadrupole Ion Traps ...... 82 A. 3D Quadrupole Ion Trap ...... 84 B. Linear Quadrupole Ion Trap (LIT) ...... 97 C. Performance Trade-Offs in the Ion Trap...... 100 3. The Orbitrap ...... 103 A. Historical Aspects ...... 103 B. Operating Principles...... 103 1) Role of the C Trap in Success of the Orbitrap ...... 106 2) Figures of Merit for the Orbitrap as an m/z Analyzer...... 107 4. Transmission Quadrupoles ...... 108 A. QMF Equations of Motion ...... 109 B. The Stability Diagram...... 110 C. Characteristics of Output ...... 111 D. Spectral Skewing ...... 113 E. Performance Limitations...... 115 5. Magnetic-Sector Instruments ...... 115 A. Single-Focusing Instruments ...... 116 1) Operating Principles ...... 116 2) Magnetic Versus Scanning...... 117 3) Performance Limitations ...... 118 B. Double-Focusing Instruments...... 118 6. FTICR-MS ...... 122 A. Hardware Configuration...... 123 B. Operational Considerations ...... 126 C. Representative Applications ...... 127 7. Ion Mobility Spectrometry (IMS) ...... 128 A. Operating Principles...... 128 B. FAIMS...... 129 C. Applications...... 130 IV. Calibration of the m/z Scale ...... 131 1. Electron Ionization ...... 132 2. Chemical Ionization ...... 133 3. Electrospray Ionization and APCI Techniques ...... 134 4. MALDI...... 135 V. Ion Detectors ...... 136 1. General Considerations...... 136 2. Types of Detectors ...... 137 A. Faraday Cup ...... 138 B. Electron Multiplier ...... 139 1) Discrete-Dynode Version ...... 139 2) Continuous-Dynode Version...... 140 C. Negative-Ion Detection ...... 142 D. Post-Acceleration Detection and Detection of High-Mass Ions ...... 143 E. Channel Electron Multiplier Array (CEMA)...... 144 viii Contents

F. Electro-Optical Ion Detection ...... 144 G. The Daly Detector...... 145 H. Cryogenic Detectors ...... 146 I. Ion Detection in FTMS ...... 147 VI. Vacuum Systems...... 147 1. Introduction...... 147 2. Definitions...... 148 3. Pressure Gauges ...... 150 A. Thermal-Conductivity Gauges ...... 150 B. Pirani Gauge...... 150 C. Thermocouple Gauges ...... 151 4. Ionization Gauges...... 151 A. Hot-Cathode Gauge...... 151 B. Cold-Cathode Gauge...... 151 5. Types of Pumps ...... 152 A. Mechanical Pumps (Low Vacuum) ...... 152 1) Rotary Vane Pumps ...... 153 2) Scroll Pumps ...... 154 3) Roots Pumps...... 155 4) Diaphram Pumps ...... 156 B. High Vacuum ...... 156 1) Turbomolecular Pumps...... 157 2) Oil Diffusion Pumps...... 160 3) Sputter-Ion Pumps (Nonregeneratable Getter Pumps)...... 162

Chapter 3 Mass Spectrometry/Mass Spectrometry...... 173 I. Introduction ...... 175 1. History and the Evolution of the Technique...... 175 2. Concept and Definitions ...... 176 3. Nomenclature...... 177 II. Ion Dissociation...... 179 1. Metastable Ions ...... 179 2. Collisionally Activated Dissociation ...... 180 3. Electron Capture Dissociation ...... 181 4. Electron-Transfer Dissociation ...... 181 5. Illustrative Example of Qualitative Analysis by MS/MS ...... 184 III. Instrumentation for MS/MS ...... 184 1. Tandem-in-Space Mass Spectrometry (MS/MS)...... 186 A. Triple-Quadrupole Mass Spectrometer ...... 187 B. Q-TOF Hybrid Mass Spectrometer...... 188 C. TOF-TOF Mass Spectrometer...... 190 D. BEqQ Hybrid Mass Spectrometer...... 190 E. Double-Focusing Instrument...... 191 2. Tandem-in-Time Mass Spectrometry...... 192 IV. Specialized Techniques and Applications...... 196 1. In-Source CAD ...... 196 2. CAD in Conjunction with Soft Ionization...... 197 A. Data-Dependent Acquisition ...... 199

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3. Selected Reaction Monitoring...... 199 A. Illustrative Example Showing that SRM Has a Higher Specificity than SIM in Spite of a Lower Signal Strength ...... 199 B. An Example Comparing the Specificity of SRM and SIM in the Context of Analyzing a Biological Sample for a Drug Metabolite....201 4. Precursor-Ion Analysis ...... 204 5. Neutral-Loss (Common Neutral-Loss) Analysis ...... 205 6. Ion/Molecule Reactions ...... 206 7. Hybrid Instrumentation for MS/MS and Ion Mobility Spectrometry (IMS) ...... 206 V. Analyte Identification from MS/MS Data ...... 208 1. Introduction...... 208 2. Identifying an Unknown Using a Product-Ion Mass Spectrum ...... 209 3. Similarities between EI and Product-Ion Mass Spectra ...... 214 4. Another Way of Using Substructure Identification...... 215 5. Searching of Product-Ion Spectra against Standardized Databases ....218 VI. Concluding Remarks about MS/MS...... 220

Chapter 4 Inlet Systems ...... 229 I. Introduction ...... 231 II. Batch Inlets ...... 232 1. Heated Reservoir Inlet...... 232 2. Direct Inlet Probe (DIP) ...... 233 A. The Chromatoprobe...... 235 3. Direct Exposure Probe (Desorption Chemical Ionization, DCI)...... 236 4. Pyrolysis ...... 238 III. Continuous Inlets ...... 239 1. Membrane Introduction MS (MIMS) ...... 239 2. Supercritical Fluid Chromatography (SFC) ...... 240 3. Electrophoretic Inlet...... 242 IV. Ionization Inlet Systems ...... 244 1. Direct Analysis in Real Time (DART) ...... 245 2. Desorption Electrospray Ionization (DESI) ...... 247 3. Desorption Atmospheric Pressure Chemical Ionization (DAPCI)...... 249 V. Speciality Interfaces...... 249 1. Selected Ion Flow Tube Mass Spectrometry (SIFTMS)...... 249 2. Fast Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry (LSIMS)...... 250 3. Chemical Reaction Interface Mass Spectrometry (CRIMS) ...... 252 4. Inductively Coupled Plasma Mass Spectrometry (ICPMS) ...... 253 A. Hardware Configuration...... 254 B. Operational Considerations ...... 254 C. Electrothermal Vaporization...... 255 D. Laser Ablation ...... 255 E. Speciation ...... 256 F. Summary...... 256 VI. Final Statement...... 257

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Chapter 5 Strategies for Data Interpretation (Other than Fragmentation) ...... 267 I. Introduction ...... 269 II. Some Important Definitions ...... 271 III. Possible Information That Can Be Obtained from the Mass Spectrum...... 271 IV. Elemental Composition of an Ion and the Ratios of Its Isotope Peaks ...... 273 1. Definition of Terms Related to the Matter of Mass Spectrometry ...... 273 2. Nitrogen Rule ...... 275 3. Elemental Composition of an Ion Based on the Ratio of Isotope Peak Intensities ...... 276 A. Isotope Peak Patterns Used to Determine the Elemental Composition of Ions ...... 276 B. Isotope Peak Patterns for Ions Containing Various Combinations of Br/Cl...... 279 C. Constraint on the Number of Atoms Allowed for a Given Element ...... 281 D. Relationship of the Charge State of an Ion and the Spacing of the Corresponding Isotope Peaks ...... 281 1) Ions of High Mass-to-Charge Ratio...... 282 E. Steps to Assigning an Elemental Composition Based on Isotope Peak Intensities ...... 283 F. Validating the Putative Elemental Composition of an Ion ...... 284 G. An Illustrative Example of the Use of Isotope Peak Ratios to Determine an Elemental Composition ...... 285 H. Potential Problems Arising from Adjacent Peaks ...... 291 4. Elemental Composition as a Function of an Accurate Determination of the m/z Value of a Mass Spectral Peak...... 293 A. Appearance of Mass Spectra of High-m/z Value Ions ...... 295 5. Using EI Data to Identify Unknowns Detected During Analysis by LC/MS ...... 297 6. Does the Result Make Sense?...... 299 V. Identifying the Mass of an Analyte ...... 302 1. Recognition of the Peak Representing the Molecular Ion in EI...... 304 A. Reasonable Losses from the Molecular Ion in EI ...... 305 2. Recognition of the Protonated Molecule (MH) in Soft Ionization...... 305 A. Probable Adducts Observed in the Mass Spectrum Produced by Soft Ionization ...... 306 3. Recognition of the Deprotonated Molecule ([M  H]  ) Peak in Soft Ionization...... 306 VI. Recognition of Spurious Peaks in the Mass Spectrum...... 307 1. Noise Spikes ...... 307 2. Peaks Corresponding to Contaminants in GC/MS and LC/MS...... 307 A. The Phthalate Peak ...... 307 B. GC Column Bleed...... 308 C. Cluster Ions...... 308 VII. Obtaining Structural Information from the Mass Spectrum ...... 308

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Chapter 6 Electron Ionization ...... 315 I. Introduction ...... 317 II. Ionization Process...... 317 III. Strategy for Data Interpretation ...... 321 1. Assumptions...... 321 2. The Ionization Process...... 321 IV. Types of Fragmentation Pathways...... 328 1. Sigma-Bond Cleavage ...... 330 2. Homolytic or Radical-Site-Driven Cleavage ...... 333 3. Heterolytic or Charge-Site-Driven Cleavage ...... 335 4. Rearrangements...... 337 A. Hydrogen-Shift Rearrangements ...... 338 B. Hydride-Shift Rearrangements ...... 342 V. Representative Fragmentations (Spectra) of Classes of Compounds...... 344 1. Hydrocarbons ...... 345 A. Saturated Hydrocarbons ...... 345 1) Straight-Chain Hydrocarbons ...... 345 2) Branched Hydrocarbons...... 348 3) Cyclic Hydrocarbons...... 351 B. Unsaturated ...... 353 C. Aromatic...... 355 2. Alkyl Halides ...... 363 3. Oxygen-Containing Compounds ...... 368 A. Aliphatic Alcohols...... 368 B. Aliphatic Ethers ...... 372 C. Aromatic Alcohols ...... 376 D. Cyclic Ethers ...... 381 E. Ketones and Aldehydes ...... 381 F. Aliphatic Acids and Esters ...... 392 G. Aromatic Acids and Esters...... 402 4. Nitrogen-Containing Compounds ...... 405 A. Aliphatic Amines ...... 405 B. Aromatic Compounds Containing Atoms of Nitrogen ...... 414 C. Heterocyclic Nitrogen-Containing Compounds...... 419 D. Nitro Compounds...... 419 E. Concluding Remarks on the Mass Spectra of Nitrogen-Containing Compounds ...... 420 5. Multiple Heteroatoms or Heteroatoms and a Double Bond...... 421 6. Trimethylsilyl Derivative...... 422 7. Determining the Location of Double Bonds...... 429 VI. Library Searches and EI Mass Spectral Databases ...... 433 1. Databases ...... 433 2. Library Search Programs ...... 435 3. What To Do When the Spectrum of the Unknown is Not in the Database(s)...... 439 4. Searching Multiple Databases...... 440 5. Database Size and Quality...... 440 6. Concluding Remarks on the NIST Mass Spectral Search Program .....441 7. Mass Spectral Database Update...... 442 VII. Summary of Interpretation of EI Mass Spectra ...... 442 xii Contents

Chapter 7 Chemical Ionization...... 449 I. Introduction ...... 451 II. Description of the Chemical Ionization Source...... 454 III. Production of Reagent Ions from Various Reagent Gases...... 455 IV. Positive-Ion Formation Under CI ...... 457 1. Fundamentals...... 457 2. Practical Consideration of Proton Affinity in CI...... 460 3. Selective Ionization...... 461 4. Fragmentation ...... 461 V. Negative-Ion Formation under CI ...... 464 1. True Negative Chemical Ionization ...... 464 2. Resonant Electron Capture Negative Ionization...... 465 VI. Data Interpretation and Systematic Studies of CI...... 469 VII. Ionization by Charge Exchange...... 470 1. Mechanism of Ionization...... 470 2. Fragmentation and Appearance of Mass Spectra...... 471 VIII. Atmospheric Pressure Chemical Ionization ...... 471 IX. Desorption Chemical Ionization ...... 472 X. General Applications...... 474 XI. Concluding Remarks ...... 477

Chapter 8 Electrospray Ionization ...... 485 I. Introduction...... 487 II. Operating Principles ...... 487 III. Appearance of ESI Mass Spectra and Data Interpretation...... 490 IV. ESI with an m/z Analyzer of High Resolving Power...... 493 V. Conventional ESI Source Interface ...... 494 VI. Nanoelectrospray and Microelectrospray Ionization...... 494 VII. Desorption Electrospray Ionization (DESI) ...... 496 VIII. Effect of Composition and Flow Rate of an Analyte Solution ...... 499 IX. Special Applications ...... 500 1. Direct Analysis of Ions in Solution by ESI...... 500 2. Cold-Spray Ionization ...... 501 3. Negative-Ion Detection...... 501 4. Secondary Electrospray Ionization (SESI) ...... 502 5. Kinetic Measurements of Chemical Reactions...... 502 6. ESI Generation of Ions for Ancillary Experiments ...... 502 X. General Applications of ESI ...... 503

Chapter 9 MALDI ...... 519 I. Historical Perspective and Introduction...... 521 II. Operating Principles ...... 521 1. The Matrix ...... 521 2. The Laser, m/z Analyzer, and Representative Mass Spectra ...... 525 3. The Ionization Process ...... 529 4. High-Pressure (HP) MALDI and Atmospheric Pressure (AP) MALDI...... 533

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III. Sample Handling...... 535 1. Sample Preparation of the Conventional Plate...... 535 2. The Problem of Analyte Solubility ...... 537 3. The Problem of Sample Purity...... 537 4. On-Probe Sample Purification and/or Modification ...... 538 A. Polymer-Modified Surfaces ...... 538 B. Affinity Surfaces...... 540 5. Direct Analysis from Gels...... 541 6. Hydrogen/Deuterium Exchange...... 542 IV. Special Instrumental Techniques ...... 542 1. Post-Source Decay (PSD) ...... 542 2. Ion Excitation ...... 544 3. Delayed Extraction (DE)...... 545 4. Desorption Ionization On Silicon (DIOS)...... 546 5. Tissue Profiling or Imaging...... 547 V. Representative Applications ...... 549 1. Proteins and Peptides ...... 549 2. Microbes ...... 549 3. Biomarkers ...... 550 4. Synthetic Polymers...... 550 5. Small Molecules ...... 551 6. Quantitation ...... 552 7. Combined with Liquid Chromatography...... 553

Chapter 10 Gas Chromatography/Mass Spectrometry...... 571 I. Introduction ...... 573 II. Introduction to GC...... 575 1. Basic Types of Injectors ...... 582 2. Injection Considerations and Syringe Handling...... 583 3. Syringeless Modes of Sample Injection for Fast GC...... 585 III. Sample Handling ...... 585 1. Proper Sample Container...... 585 2. Analyte Isolation and Purification ...... 587 3. Derivative Formation ...... 587 A. Silyl Derivatives...... 588 B. Esters of Carboxylic Acids ...... 588 C. Oxime Derivatives...... 589 D. Acyl Derivatives...... 590 E. Derivatives for Characterizing Double Bonds ...... 590 IV. Instrument Requirements for GC/MS ...... 590 1. Operating Pressures ...... 590 2. Typical Parameters for a Conventional GC-MS Interface ...... 593 3. Supersonic Molecular Beam Interface for GC/MS ...... 594 4. Open-Split Interface ...... 596 5. Molecular Separators ...... 597 A. Jet-Orifice Separator...... 597 B. Membrane Separator...... 598 6. Inertness of Materials in the Interface ...... 599

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V. Operational Considerations ...... 601 1. Spectral Skewing...... 601 2. Background/Bleed ...... 602 3. The Need for Rapid Acquisition of Mass Spectra ...... 604 A. Performance Trade-Offs of Conventional Instruments for GC/MS...... 605 B. Time-Array Detection...... 605 4. Selected Ion Monitoring (SIM)...... 606 A. Definition and Nomenclature ...... 606 B. Development of the Technique...... 607 C. Qualitative Example of SIM ...... 607 D. Quantitative Example of SIM ...... 609 E. Mechanics of Ion Monitoring...... 613 1) Adjustment of the Mass Scale...... 613 2) Mass Range ...... 613 3) Magnetic Mass Spectrometer ...... 613 4) Transmission Quadrupole Mass Spectrometer...... 614 5) Number of Ion Currents (Masses)...... 614 F. Programmable SIM...... 614 G. SIM at High Resolving Power ...... 615 VI. Sources of Error ...... 616 1. Errors Relating to Equipment or Procedure ...... 616 2. Errors Relating to Contamination ...... 617 3. Sources of Interference...... 618 4. Dealing with Background in a Mass Spectrum...... 618 A. AMDIS (Automated Mass spectral Deconvolution and Identification System)...... 622 B. Other Software Techniques ...... 629 VII. Representative Applications of GC/MS...... 631 VIII. Special Techniques...... 631 1. Purge and Trap ...... 631 2. Thermal Desorption...... 632

Chapter 11 Liquid Chromatography/Mass Spectrometry ...... 639 I. Introduction ...... 641 II. Historical Milestones in the Development of the Interface ...... 642 1. Introduction...... 642 2. The Direct Inlet ...... 642 3. The Moving-Belt Interface ...... 644 4. The Thermospray Interface...... 644 5. Continuous-Flow FAB ...... 646 III. Currently Viable Versions of the Interface...... 647 1. Atmospheric Pressure Ionization...... 647 A. Electrospray Ionization Interface...... 647 1) Optimization for Analyses by HPLC ...... 648 2) Capillary Electrophoresis Interface...... 650 B. APCI Interface ...... 650

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C. APPI Interface...... 654 1) Operating Principles of APPI...... 654 2) Operating Mechanics for APPI...... 656 3) Signal Suppression ...... 657 4) Applications of APPI...... 658 2. Particle Beam Interface...... 659 3. Electron Ionization and LC/MS...... 661 IV. Special Operation of LC under MS Conditions ...... 661 1. Effects of Mobile-Phase Composition ...... 661 A. Signal Suppression ...... 662 B. Use of Internal Standards in the Face of Signal Suppression ...... 663 C. Adjusting the Chromatography in the Face of Signal Suppression during LC/MS...... 663 D. Ion Pairing and Signal Suppression...... 663 E. Influence of the Type and the Nature of LC Buffer ...... 665 F. Influence of Solvent Composition on the ESI Signal...... 665 G. Adduct Formation ...... 667 H. Spectral Interference ...... 668 I. System Compromise ...... 668 2. Differences in Method Development for ESI vs APCI ...... 672 V. Applications ...... 674 1. Attention to High Throughput ...... 676

Chapter 12 Analysis of Proteins and Other Biopolymers ...... 689 I. Introduction ...... 691 II. Proteins ...... 691 1. Sequencing...... 693 A. Nomenclature and Fragmentation in Sequencing of Peptides ...... 693 1) Nomenclature...... 693 2) Fragmentation ...... 695 B. Strategy for Deducing Amino Acid Sequence via CAD of Peptides...... 701 1) An Illustrative Example...... 702 2) Possible Pitfalls in Interpretation ...... 705 3) Search for Confirming Ions ...... 706 4) Ladder Sequencing ...... 707 2. Mass Mapping ...... 709 A. Peptide Mass Fingerprinting ...... 709 B. De novo Sequencing...... 710 C. Sequence Tagging...... 710 D. Sequest...... 710 E. Evaluation of Hits in Automated Searches ...... 711 F. Data-Dependent Analysis by Mass Spectrometry ...... 712 3. Post-Translational Modifications ...... 712 A. Recognition of Sites of Protein Phosphorylation...... 714 1) An Illustrative Example...... 715 2) Selective Capture and Detection of Phosphopeptides ...... 718 3) Chemical Modification of Phosphorylation Sites ...... 719

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B. Recognition of Sites of Sulfation...... 722 C. Recognition of Sites of Glycosylation...... 723 D. Acetylation of Lysine ...... 724 E. Cysteine Status in Proteins...... 726 1) Are There Any Disulfide Bonds? ...... 726 2) Which Cysteines Are Free? ...... 727 3) What Is the Linkage of Cysteines in the Disulfide Bonds? ...... 727 (A) Conventional Proteolytic Mass Mapping of Disulfides ...... 727 (B) Cyanylation-Based Mass Mapping of Disulfides...... 730 F. Recognition of Ubiquinated Proteins...... 734 G. Other Types of Modifications ...... 735 4. Quantitation in Proteomics ...... 735 A. ICATs...... 735 1) Operating Principles ...... 735 2) Illustrative Example of the ICAT Approach ...... 737 3) Analogous to ICAT Methodologies...... 740 B. Alternative Stable Isotope-Based Methodologies ...... 740 C. Related Methodologies ...... 743 5. “Top-Down” Strategies of Analysis...... 744 A. Instrumentation and Fragmentation Requirements ...... 744 B. Electron Capture Dissociation (ECD) ...... 749 C. Electron-Transfer Dissociation (ETD)...... 752 D. Applications...... 753 6. Noncovalent Interactions...... 753 7. Folding and Unfolding ...... 756 8. Applications ...... 759 III. Oligonucleotides ...... 760 1. Analytical Considerations ...... 760 2. Sequencing ...... 761 A. Nomenclature ...... 761 B. Algorithm for Data Interpretation...... 763 3. Applications ...... 764 IV. Carbohydrates ...... 765 1. Analytical Considerations ...... 765 2. Nomenclature...... 765 3. Diagnostic Fragmentation ...... 766 4. Applications ...... 769

Subject Index...... 803

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