Mass Spectrometry A Textbook, 2nd Edition Jurgen H. Gross

Mass Spectrometry A Textbook, 2nd Edition Jurgen H. Gross Mass Spectrometry A Textbook, 2nd Edition Jurgen H. Gross Mass Spectrometry A Textbook, 2nd Edition Jurgen H. Gross Mass Spectrometry A Textbook, 2nd Edition Jurgen H. Gross Mass Spectrometry A Textbook, 2nd Edition Jurgen H. Gross Mass Spectrometry A Textbook, 2nd Edition Jurgen H. Gross

Second Edition

 Springer-Verlag Berlin Heidelberg 2004, 2011

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DOI 10.1007/978-3-642-10711-5

Springer Heidelberg Dordrecht London New York

Shortly after having graduated in 1966 and just employed as a research assistant in

a protein chemistry laboratory, my very first contact with mass spectrometry pened when I stumbled on a paper by Michael Barber, the later discoverer of fast atom bombardment (FAB) Together with a French group he had determined the covalent structure of an almost 1.4 kDa complex peptidolipid called fortuitine by using mass spectrometry Fascinated by this to me unknown technique, I felt that

hap-MS would be a future key analytical method in protein studies At that time, the only ionization method available was electron ionization, which required a sample

to be in the gaseous state in the ion source Therefore most mass spectrometric analyses were dealing with small organic molecules – and peptides and proteins were not volatile Fortuitine was a very fortuitous sample, because it was naturally derivatized with the consequence that it could be volatilized into the ion source Nevertheless, I went into mass spectrometry My first mass spectrometer was in-stalled in our laboratory in 1968 Mass spectrometers at that time were complex fully manually operated instruments most of them magnetic/electrostatic sector in-struments, and the operator needed to know the instrument well in order to avoid catastrophes by opening wrong valves at the wrong moment Spectra were re-corded on UV paper with a galvanometer recorder or on photographic plates and mass assignment was performed manually During the 1970s a number of new ionization methods and mass analyzers became available These included ioniza-tion by chemical ionization and by field ionization/desorption as well as mass analyses by quadrupoles and ion traps Computers became available for data ac-quisition and mass assignment Life became easier but the requirement for volatile samples was still there

The 1980s revolutionized the possibilities for mass spectrometric analysis In the early half of the decade introduction of FAB and commercialization of the 10 years earlier developed plasma desorption mass spectrometry allowed for analyses

of nonvolatile samples such as peptides, proteins, and nucleic acids The first commercial fully automated mass spectrometer, the BioIon plasma desorption mass spectrometer, became available and the time-of-flight analyzer, which had unlimited mass range, was revived Late in the decade the two new and now dominating ionization methods electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) were introduced These two ionization meth-ods opened a new era for mass spectrometry Now all the large nonvolatile bio-logical molecules could be analyzed Till then GC-MS had been extensively used for analysis of complex mixtures in environmental and clinical sciences, but due

to its nature it was limited to small volatile molecules ESI made coupling of LC with MS possible allowing for entirely new applications of mass spectrometry Proteomics now became a big move forward with mass spectrometry as the key analytical tool Thousands of scientists took up mass spectrometric analysis and

Fourier transformation, has become standard in advanced biological research ratories Biological mass spectrometry and especially analysis of proteins and pro-teomics now dominate mass spectrometry conferences and mass spectrometry has

labo-a strong position in biologiclabo-al conferences, where these subjects ten yelabo-ars elabo-arlier were only marginally present

What are the consequences of this development? For me, having tried to get mass spectrometry into protein science for more than 40 years it is of course en-couraging Mass spectrometry is without any doubt now the most versatile ana-lytical technique available It is used in a wide variety of areas from inorganic, nu-clear chemistry, and geochemistry over organic chemistry, environmental analyses, clinical chemistry, to molecular and cell biology Online separation of complex mixtures is possible using either GC-MS or LC-MS Almost all commer-cial instruments are highly automated However, this development also rises seri-ous concerns Many of the new users consider the mass spectrometer as a black box where they put in the sample in one end and get a result from the computer in the other end They do not or only marginally understand the principles in their in-strument and rarely look at the raw data They are satisfied with computer prints with lists of identified compounds Sample preparation often follows standard pro-tocols and the understanding of the need for optimized sample preparation for each analytical task is often ignored As a result, a considerable amount of the data obtained are questionable either due to poor sample preparation, poor instrument performance, or suboptimal use of the instruments It is my wish that the new gen-eration of mass spectrometry users will spend time to understand their instruments and the requirements for optimal preparation of the samples and it is my hope that this book will be read by many of them so that they can use their techniques to the best of the equipment’s potential

Odense, 2010

Peter Roepstorff

Department of Biochemistry and Molecular Biology

University of Southern Denmark

To all readers of the first edition of Mass Spectrometry – A Textbook I would like

to express my deepest gratitude Without their interest in wanting to learn more about mass spectrometry by use of this book, all the efforts in writing it would have been a mere waste of time, and moreover, without their demand for updates, there would be no next edition I would also like to thank the instructors all over the world who adopted and recommended this book for their own mass spectrome-try courses

Preparing the second edition of Mass Spectrometry – A Textbook was not an

easy task The years have witnessed a flood of innovations and detailed edge of interrelationships that were previously hardly understood The time be-tween the editions may have appeared a bit long for many eager scholars But the author has used the time effectively to improve and update the entire contents, hopefully to the benefit of all who have been patiently bearing with me in antici-pation

knowl-So, what’s new? The book now comprises fifteen instead of twelve chapters, each of them headed by essential “Learning Objectives” Chapter 9 inserts meth-ods of ion activation such as CID, ECD, ETD, and IRMPD closely related to the instrumental approaches to tandem mass spectrometry A second additional chap-ter deals with sampling and ion generation from surfaces under ambient conditions

as afforded by DART and DESI, to name the most relevant methods Finally, a new chapter on inorganic mass spectrometry has been added, for one, to include element speciation that bridges the gap between biomedical and trace elemental analysis and, also, to open a perspective extending beyond the key topics of this book The chapter on instrumentation has been significantly expanded to cover or-bitrap, linear ion traps, TOF/TOF, FT-ICR, and the ever-changing hybrid instru-ments including IMS-MS systems More detailed attention is drawn to applica-tions regarding biopolymers, especially in those chapters dealing with MALDI and ESI

Overall, the book has been expanded by more than 200 pages No chapter has remained untouched Numerous passages have been rewritten to improve the clar-ity of explanations while keeping them short and concise Care has been taken not only to explain how, but also to why things are done a certain way Several schemes have been added to clarify interrelationships between different tech-niques Tables compiling data for general reference where transferred to the ex-panded appendix The book’s website has been updated providing new exercises and supplementary material (http://www.ms-textbook.com)

for checking Chap 2 (Principles of Ionization and Ion Dissociation), Alexander Makarov, Thermo Fisher Scientific, Bremen (Chap 4, Instrumentation), Christoph

A Schalley, Freie Universität Berlin (Chap 9, Tandem Mass Spectrometry), Belá Paizs, German Cancer Research Center, Heidelberg (Chap 11, Matrix-Assisted

Laser Desorption/Ionization), Zoltán Takáts, Universität Gießen (Chap 13, ent Mass Spectrometry), and Detlef Günther, ETH Zürich (Chap 15, Inorganic Mass Spectrometry) Without their care and help the many new parts would not

Ambi-have reached the present level of accuracy Despite intense reviewing and reading some errors inevitably may have remained I apologize for these in ad-vance and would highly appreciate any feedback from the readership in trying to identify and correcting them

proof-I am indebted to Peter Roepstorff, Odense University, for writing the Foreword with such a personal connotation Permission to prepare this 2nd edition, alongside

my official professional duties, by A Stephen K Hashmi, Director of OCI, and Heinfried Schöler, Dean of the Faculty of Chemistry and Earth Sciences is sin-cerely acknowledged Many thanks to Doris Lang, Iris Mitsch, and Norbert Nieth, for smoothly running the routine analyses in our MS facility And again, several mass spectrometry companies are acknowledged for supplying new instrument schemes and other figures for inclusion in the 2nd edition Theodor C H Cole ac-complished a great job in polishing up my English Finally, I am immeasurably grateful to my family for their patience and solidarity in times when I had to come home late or needed to vanish on Saturdays during the writing of this book Have a good time studying, learning, and enjoying the world of massspectrometry!

When non-mass spectrometrists are talking about mass spectrometry it rather often

sounds as if they were telling a story out of Poe's Tales of Mystery and

Imagina-tion Indeed, mass spectrometry appears to be regarded as a mysterious method,

just good enough to supply some molecular weight information Unfortunately, this rumor about the dark side of analytical methods reaches students much earlier than their first contact with mass spectrometry Possibly, some of this may have been bred by mass spectrometrists themselves who tended to celebrate each mass spectrum they obtained from the gigantic machines of the early days Of course, there were also those who enthusiastically started in the 1950s to develop mass spectrometry out of the domain of physics to become a new analytical tool for chemistry

Nonetheless, some oddities remain and the method which is to be introduced herein is not always straightforward and easy If you had asked me, the author, just after having finished my introductory course whether mass spectrometry would become my preferred area of work, I surely would have strongly denied

On the other hand, J J Veith's mass spectrometry laboratory at Darmstadt sity was bright and clean, had no noxious odors, and thus presented a nice contrast

Univer-to a preparative organic chemistry laboraUniver-tory Numerous stainless steel flanges and electronics cabinets were tempting to be explored and – whoops – infected me with CMSD (chronic mass spectrometry disease) Staying with Veith's group slowly transformed me into a mass spectrometrist Inspiring books such as

Fundamental Aspects of Organic Mass Spectrometry or Metastable Ions, out of

stock even in those days, did help me very much during my metamorphosis ing completed my doctoral thesis on fragmentation pathways of isolated immo-nium ions in the gas phase, I assumed my current position Since 1994, I have been head of the mass spectrometry laboratory at the Chemistry Department of Heidelberg University where I teach introductory courses and seminars on mass spectrometry

Hav-When students ask what books to read on mass spectrometry, there are various excellent monographs, but the ideal textbook still seemed to be missing – at least

in my opinion Finally, encouraged by many people including P Enders, Verlag Heidelberg, two years of writing began

Springer-The present volume would not have its actual status without the critical reviews

of the chapters by leading experts in the field Their thorough corrections, marks, and comments were essential Therefore, P Enders, Springer-Verlag Hei-

re-delberg (Introduction), J Grotemeyer, University of Kiel (Gas Phase Ion

Chemis-try), S Giesa, Bayer Industry Services, Leverkusen (Isotopes), J Franzen, Bruker

gratefully acknowledged for sending large collections of schemes and photographs for use in this book The author wishes to express his thanks to those scientists, many of them from the University of Heidelberg, who generously allowed to use material from their actual research as examples and to those publishers, who granted the numerous copyrights for use of figures from their publications The generous permission of the National Institute of Standards and Technology (G Mallard, J Sauerwein) to use a large set of electron ionization mass spectra from the NIST/EPA/NIH Mass Spectral Library is also gratefully acknowledged

My thanks are extended to the staff of my facility (N Nieth, A Seith, B Flock) for their efforts and to the staff of the local libraries for their friendly support I am indebted to the former director of our institute (R Gleiter) and to the former dean

of our faculty (R N Lichtenthaler) for permission to write a book besides my ficial duties

of-Despite all efforts, some errors or misleading passages will still have remained Mistakes are an attribute that make us human, but unfortunately, they do not con-tribute to the scientific or educational value of a textbook Therefore, please do not hesitate to report errors to me or to drop a line of comment in order to allow for corrections in a future edition

Hopefully, Mass Spectrometry – A Textbook will introduce you to the many

facets of mass spectrometry and will satisfy your expectations

Table of Contents XI

1 Introduction 1

Learning Objectives 1

1.1 Aims and Scope 3

1.1.1 Filling the Black Box 5

1.2 What Is Mass Spectrometry? 5

1.2.1 Mass Spectrometry 6

1.2.2 Mass Spectrometer 7

1.2.3 Mass Scale 8

1.2.4 Mass Spectrum 9

1.3 Ion Chromatograms 11

1.4 Performance of Mass Spectrometers 13

1.4.1 Sensitivity 13

1.4.2 Detection Limit 14

1.4.3 Signal-to-Noise Ratio 14

1.5 Terminology – General Aspects 15

1.5.1 Basic Terminology in Describing Mass Spectra 16

1.6 Units, Physical Quantities, and Physical Constants 17

References 17

2 Principles of Ionization and Ion Dissociation 21

Learning Objectives 21

2.1 Gas Phase Ionization by Energetic Electrons 21

2.1.1 Formation of Ions 22

2.1.2 Processes Accompanying Electron Ionization 23

2.1.3 Ions Generated by Penning Ionization 24

2.1.4 Ionization Energy 25

2.1.5 Ionization Energy and Charge-Localization 25

2.2 Vertical Transitions 27

2.3 Ionization Efficiency and Ionization Cross Section 29

2.5.5 Reacting Ions Described by k(E) Functions 40

2.5.6 Direct Cleavages and Rearrangement Fragmentations 40

2.6 Time Scale of Events 42

2.6.1 Stable, Metastable, and Unstable Ions 43

2.6.2 Time Scale of Ion Storage Devices 44

2.7 Internal Energy – Practical Implications 45

2.8 Reverse Reactions and Kinetic Energy Release 46

2.8.1 Activation Energy of the Reverse Reaction 46

2.8.2 Kinetic Energy Release 48

2.8.3 Energy Partitioning 49

2.9 Isotope Effects 49

2.9.1 Primary Kinetic Isotope Effects 50

2.9.2 Measurement of Isotope Effects 51

2.9.3 Secondary Kinetic Isotope Effects 53

2.10 Determination of Ionization Energies 54

2.10.1 Conventional Determination of Ionization Energies 54

2.10.2 Improved IE Accuracy from Data Post-Processing 54

2.10.3 IE Accuracy – Experimental Improvements 55

2.10.4 Photoionization Processes 55

2.11 Determining the Appearance Energies 58

2.11.1 Kinetic Shift 58

2.11.2 Breakdown Graphs 59

2.12 Gas Phase Basicity and Proton Affinity 61

References 62

3 Isotopic Composition and Accurate Mass 67

Learning Objectives 67

3.1 Isotopic Classification of the Elements 67

3.1.1 Monoisotopic Elements 68

3.1.2 Di-isotopic Elements 68

3.1.3 Polyisotopic Elements 69

3.1.4 Representation of Isotopic Abundances 69

3.1.5 Calculation of Atomic, Molecular, and Ionic Mass 71

3.1.6 Natural Variations in Relative Atomic Mass 73

3.2 Calculation of Isotopic Distributions 74

3.2.1 Carbon: An X+1 Element 74

3.2.2 Terms Related to Isotopic Composition 77

3.2.3 Binomial Approach 77

3.2.4 Halogens 78

3.2.5 Combinations of Carbon and Halogens 80

3.2.6 Polynomial Approach 81

3.2.7 Oxygen, Silicon, and Sulfur 81

3.2.8 Polyisotopic Elements 84

3.2.9 Practical Aspects of Isotopic Patterns 84

3.2.10 Bookkeeping with Isotopic Patterns in Mass Spectra 85

3.2.11 Information from Complex Isotopic Patterns 86

3.3 Isotopic Enrichment and Isotopic Labeling 87

3.3.1 Isotopic Enrichment 87

3.3.2 Isotopic Labeling 88

3.4 Resolution and Resolving Power 88

3.4.1 Definitions 88

3.4.2 Resolution and its Experimental Determination 90

3.4.3 Resolving Power and its Effect on Relative Peak Intensity 91

3.5 Accurate Mass 92

3.5.1 Exact Mass and Molecular Formulas 92

3.5.2 Mass Defect 93

3.5.3 Mass Accuracy 95

3.5.4 Accuracy and Precision 96

3.5.5 Mass Accuracy and the Determination of Molecular Formulas 97

3.5.6 Extreme Mass Accuracy – Special Considerations 98

3.6 Applied High-Resolution Mass Spectrometry 99

3.6.1 External Mass Calibration 99

3.6.2 Internal Mass Calibration 101

3.6.3 Compiling Mass Reference Lists 103

3.6.4 Specification of Mass Accuracy 104

3.6.5 Deltamass 104

3.6.6 Kendrick Mass Scale 105

3.6.7 Van Krevelen Diagrams 106

3.7 Resolution Interacting with Isotopic Patterns 107

3.7.1 Multiple Isotopic Compositions at Very High Resolution 107

3.7.2 Isotopologs and Accurate Mass 110

3.7.3 Large Molecules – Isotopic Patterns at Sufficient Resolution 110

3.7.4 Large Molecules – Isotopic Patterns at Low Resolution 112

3.8 Charge State and Interaction with Isotopic Patterns 112

References 114

4 Instrumentation 117

Learning Objectives 117

4.1 How to Create a Beam of Ions 119

4.2 Time-of-Flight Instruments 120

4.2.1 Time-of-Flight – Basic Principles 120

4.2.2 TOF Instruments – Velocity of Ions and Time-of-Flight 121

4.2.3 Linear Time-of-Flight Analyzer 123

4.3.2 Principle of the Magnetic Sector 136

4.3.3 Focusing Action of the Magnetic Field 138

4.3.4 Double-Focusing Sector Instruments 139

4.3.5 Geometries of Double-Focusing Sector Instruments 141

4.3.6 Adjusting the Resolving Power of a Sector Instrument 143

4.3.7 Innovations in Sector Instruments 144

4.4 Linear Quadrupole Instruments 146

4.4.1 Introduction 146

4.4.2 The Linear Quadrupole 147

4.4.3 Resolving Power of Linear Quadrupoles 151

4.4.4 RF-Only Quadrupoles, Hexapoles, and Octopoles 152

4.5 Linear Quadrupole Ion Traps 155

4.5.1 Linear RF-Only Multipole Ion Traps 155

4.5.2 Mass-Analyzing Linear Quadrupole Ion Trap with Axial Ejection 158 4.5.3 Mass-Analyzing Linear Ion Trap with Radial Ejection 160

4.6 Three-Dimensional Quadrupole Ion Trap 164

4.6.1 Introduction 164

4.6.2 The Quadrupole Ion Trap 164

4.6.3 Visualization of Ion Motion in the Ion Trap 167

4.6.4 Mass-Selective Stability Mode 168

4.6.5 Mass-Selective Instability Mode 168

4.6.6 Resonant Ejection 169

4.6.7 Axial Modulation and Automatic Gain Control 170

4.6.8 Nonlinear Resonances 171

4.6.9 Digital Waveform Quadrupole Ion Trap 172

4.6.10 External Ion Sources for the Quadrupole Ion Trap 173

4.7 Fourier Transform Ion Cyclotron Resonance 174

4.7.1 Ion Cyclotron Resonance 174

4.7.2 Ion Cyclotron Motion 174

4.7.3 Cyclotron Motion – Excitation and Detection 175

4.7.4 Cyclotron Frequency Bandwidth and Energy-Time Uncertainty 177

4.7.5 Fourier Transform – Basic Properties 179

4.7.6 Nyquist Criterion 181

4.7.7 Excitation Modes in FT-ICR-MS 182

4.7.8 Axial Trapping and Design of ICR Cells 183

4.7.9 Magnetron Motion and Reduced Cyclotron Frequency 184

4.7.10 Detection and Accuracy in FT-ICR-MS 186

4.7.11 FT-ICR Instruments 187

4.8 Orbitrap Analyzer 189

4.8.1 Orbitrap – Principle of Operation 189

4.8.2 Ion Detection and Resolving Power of the Orbitrap 191

4.8.3 Ion Injection into the Orbitrap 192

4.8.4 Hybridization with a Linear Quadrupole Ion Trap 193

4.9 Hybrid Instruments 194

4.9.1 Evolution of Hybrid Mass Spectrometers 196

4.9.2 Ion Mobility-Mass Spectrometry Systems 198

4.10 Detectors 202

4.10.1 Discrete Dynode Electron Multipliers 203

4.10.2 Channel Electron Multipliers 204

4.10.3 Microchannel Plates 205

4.10.4 Post-Acceleration and Conversion Dynode 206

4.10.5 Focal Plane Detectors 207

4.11 Vacuum Technology 208

4.11.1 Basic Mass Spectrometer Vacuum System 209

4.11.2 High Vacuum Pumps 209

4.12 Purchasing an Instrument 210

References 210

5 Practical Aspects of Electron Ionization 223

Learning Objectives 223

5.1 Electron Ionization Ion Sources 223

5.1.1 Layout of an Electron Ionization Ion Source 223

5.1.2 Generation of Primary Electrons 225

5.1.3 Overall Efficiency and Sensitivity of an El Ion Source 226

5.1.4 Optimization of Ion Beam Geometry 227

5.2 Sample Introduction 228

5.2.1 Reservoir or Reference Inlet System 228

5.2.2 Direct Insertion Probe 231

5.2.3 Sample Vials for Use with Direct Insertion Probes 232

5.2.4 Fractionation When Using Direct Insertion Probes 233

5.2.5 Direct Exposure Probe 235

5.3 Pyrolysis Mass Spectrometry 237

5.4 Gas Chromatograph 237

5.5 Liquid Chromatograph 238

5.6 Low-Energy Electron Ionization Mass Spectra 239

5.7 Analytes for EI 241

5.8 Mass Analyzers for EI 241

5.9 Mass Spectral Databases for EI 242

5.9.1 NIST/EPA/NIH Mass Spectral Database 243

5.9.2 Wiley Registry of Mass Spectral Data 244

5.9.3 Mass Spectral Databases – General Aspects 244

References 245

6.2.3 α-Cleavage of Nonsymmetrical Aliphatic Ketones 259

6.2.4 Acylium Ions and Carbenium Ions 260

6.2.5 α-Cleavage When Heteroatoms Belong to the Aliphatic Chain 262

6.2.6 α-Cleavage of Aliphatic Amines 262

6.2.7 Nitrogen Rule 265

6.2.8 α-Cleavage of Aliphatic Ethers and Alcohols 266

6.2.9 Charge Retention at the Heteroatom 268

6.2.10 α-Cleavage of Thioethers 269

6.2.11 α-Cleavage of Halogenated Hydrocarbons 269

6.2.12 Double α-Cleavage 271

6.2.13 Double α-Cleavage for the Identification of Regioisomers 272

6.3 Distonic Ions 273

6.3.1 Definition of Distonic Ions 273

6.3.2 Formation and Properties of Distonic Ions 274

6.3.3 Distonic Ions as Intermediates 275

6.4 Benzylic Bond Cleavage 275

6.4.1 Cleavage of the Benzylic Bond in Phenylalkanes 275

6.4.2 The Further Fate of [C6H5]+ and [C7H7]+ 277

6.4.3 Isomerization of [C7H8]+• and [C8H8]+• Ions 279

6.4.4 Rings Plus Double Bonds 280

6.5 Allylic Bond Cleavage 281

6.5.1 Cleavage of the Allylic Bond in Aliphatic Alkenes 281

6.5.2 Methods for the Localization of the Double Bond 283

6.6 Cleavage of Non-Activated Bonds 284

6.6.1 Saturated Hydrocarbons 284

6.6.2 Carbenium Ions 286

6.6.3 Very Large Hydrocarbons 287

6.6.4 Recognition of the Molecular Ion Peak 288

6.7 McLafferty Rearrangement 290

6.7.1 McL of Aldehydes and Ketones 290

6.7.2 Fragmentation of Carboxylic Acids and Their Derivatives 293

6.7.3 McL of Aromatic Hydrocarbons 296

6.7.4 McL with Double Hydrogen Transfer 297

6.8 Retro-Diels-Alder Reaction 300

6.8.1 Properties of the Retro-Diels-Alder Reaction 300

6.8.2 Influence of Positional Isomerism on the RDA Reaction 302

6.8.3 RDA Reaction in Natural Products 303

6.8.4 Widespread Occurrence of the RDA Reaction 303

6.9 Elimination of Carbon Monoxide 304

6.9.1 CO Loss from Phenols 304

6.9.2 CO and C2H2 Loss from Quinones 307

6.9.3 Fragmentation of Arylalkylethers 308

6.9.4 CO Loss from Transition Metal Carbonyl Complexes 310

6.9.5 CO Loss from Carbonyl Compounds 311

6.9.6 Differentiation Between Loss of CO, N2, and C2H4 311

6.10 Thermal Degradation vs Ion Fragmentation 312

6.10.1 Decarbonylation and Decarboxylation 312

6.10.2 Retro-Diels-Alder Reaction 312

6.10.3 Loss of H2O from Alkanols 312

6.10.4 EI Mass Spectra of Organic Salts 314

6.11 Alkene Loss from Onium Ions 315

6.11.1 McL of Onium Ions 316

6.11.2 Onium Reaction 319

6.12 Ion-Neutral Complexes 322

6.12.1 Evidence for the Existence of Ion-Neutral Complexes 322

6.12.2 Attractive Forces in Ion-Neutral Complexes 323

6.12.3 Criteria for Ion-Neutral Complexes 324

6.12.4 Ion-Neutral Complexes of Radical Ions 325

6.13 Ortho Elimination (Ortho Effect) 326

6.13.1 Ortho Elimination from Molecular Ions 327

6.13.2 Ortho Elimination from Even-Electron Ions 328

6.13.3Ortho Elimination in the Fragmentation of Nitroarenes 331

6.14 Heterocyclic Compounds 332

6.14.1 Saturated Heterocyclic Compounds 333

6.14.2 Aromatic Heterocyclic Compounds 336

6.15 Guide to the Interpretation of Mass Spectra 340

6.15.1 Summary of Rules 340

6.15.2 Systematic Approach to Mass Spectra 341

References 342

7 Chemical Ionization 351

Learning Objectives 351

7.1 Basics of Chemical Ionization 351

7.1.1 Formation of Ions in Positive-Ion Chemical Ionization 351

7.1.2 Chemical Ionization Ion Sources 352

7.1.3 Sensitivity of Chemical Ionization 353

7.1.4 Chemical Ionization Techniques and Terms 353

7.2 Protonation in Chemical Ionization 354

7.2.1 Source of Protons 354

7.2.2 Methane Reagent Gas Plasma 355

7.2.3 CH5 and Related Ions 356

7.4.3 Compound Class-Selective CE-CI 366

7.4.4 Regio- and Stereoselectivity in CE-CI 368

7.5 Negative-Ion Chemical Ionization 368

7.6 Electron Capture 370

7.6.1 Ion Formation by Electron Capture 370

7.6.2 Energetics of EC 370

7.6.3 Creating Thermal Electrons 372

7.6.4 Appearance of EC Spectra 373

7.6.5 Applications of EC 373

7.7 Desorption Chemical Ionization 374

7.8 Analytes for CI 375

References 376

8 Field Ionization and Field Desorption 381

Learning Objectives 381

8.1 Field Ionization Process 382

8.2 FI and FD Ion Sources 383

8.3 Field Emitters 385

8.3.1 Blank Metal Wires as Emitters 385

8.3.2 Activated Emitters 385

8.3.3 Emitter Temperature 386

8.3.4 Handling of Activated Emitters 387

8.4 Field Ionization Mass Spectrometry 388

8.4.1 Origin of [M+H]+ Ions in FI-MS 389

8.4.2 Multiply-Charged Ions in FI-MS 389

8.4.3 Field-Induced Dissociation 390

8.4.4 Accurate Mass FI Spectra 390

8.4.5 Coupling Gas Chromatography to FI-MS 391

8.5 FD Spectra 392

8.5.1 Ion Formation by Field Ionization in FD-MS 393

8.5.2 Desorption of Preformed Ions in FD-MS 394

8.5.3 Cluster Ion Formation in FD-MS 396

8.5.4 FD-MS of Ionic Analytes 397

8.5.5 Best Anode Temperature and Thermal Decomposition 399

8.5.6 FD-MS of Polymers 400

8.5.7 Types of Ions in FD-MS 401

8.6 Liquid Injection Field Desorption Ionization 402

8.7 General Properties of FI-MS and FD-MS 405

8.7.1 Sensitivity of FI-MS and FD-MS 405

8.7.2 Analytes and Practical Considerations for FI, FD, and LIFDI 407

8.7.3 Mass Analyzers for FI and FD 407

References 408

9 Tandem Mass Spectrometry 415

Learning Objectives 415

9.1 Concepts of Tandem Mass Spectrometry 415

9.1.1 Tandem-in-Space and Tandem-in-Time 416

9.1.2 Pictograms for MS/MS Experiments 418

9.2 Metastable Ion Dissociation 420

9.3 Collision-Induced Dissociation 420

9.3.1 Effecting Collisions in a Mass Spectrometer 420

9.3.2 Energy Transfer During Collisions 421

9.3.4 Single and Multiple Collisions in CID 424

9.3.5 Time Scale of Ion Activating Processes 426

9.4 Surface-Induced Dissociation 426

9.5 Tandem MS on TOF Instruments 427

9.5.1 Utilizing a ReTOF for Tandem MS 427

9.5.2 Curved-Field Reflectron 429

9.5.3 Tandem MS on True Tandem TOF Instruments 429

9.6 Tandem MS with Magnetic Sector Instruments 431

9.6.1 Dissociations in the FFR Preceding the Magnetic Sector 431

9.6.2 Mass-Analyzed Ion Kinetic Energy Spectra 432

9.6.3 Determination of Kinetic Energy Release 432

9.6.4 B/E = Const Linked Scan 434

9.6.5 Additional Linked Scan Functions 434

9.6.6 Multi-Sector Instruments 436

9.7 Tandem MS with Linear Quadrupole Analyzers 437

9.7.1 Triple Quadrupole Mass Spectrometers 437

9.7.2 Scan Modes for Tandem MS with Triple Quadrupole Instruments 438

9.7.3 Penta Quadrupole Instruments 438

9.8 Tandem MS with the Quadrupole Ion Trap 439

9.9 Tandem MS with Linear Quadrupole Ion Traps 443

9.9.1 Tandem MS on QqLIT Instruments 444

9.9.2 Tandem MS on LITs with Radial Ejection 444

9.10 Tandem MS with Orbitrap Instruments 445

9.10.1 Higher-Energy C-Trap Dissociation 446

9.10.2 Extended LIT-Orbitrap Hybrid Instruments 446

9.11 Tandem MS with FT-ICR Instruments – Part I 448

9.11.1 Sustained Off-Resonance Irradiation-CID in ICR Cells 448

9.12 Infrared Multiphoton Dissociation 451

9.12.1 IRMPD in QITs and LITs 452

9.13 Electron Capture Dissociation 452

9.18 Special Applications of Tandem MS 463

9.18.1 Ion–Molecule Reactions in Catalytic Studies 464

9.18.2 Gas Phase Hydrogen–Deuterium Exchange 464

9.18.3 Determination of Gas Phase Basicities and Proton Affinities 466

9.18.4 Neutralization-Reionization Mass Spectrometry 467

References 468

10 Fast Atom Bombardment 479

Learning Objectives 479

10.1 Ion Sources for FAB and LSIMS 480

10.1.1 FAB Ion Sources 480

10.1.2 LSIMS Ion Sources 482

10.1.3 FAB Probes 482

10.2 Ion Formation in FAB and LSIMS 483

10.2.1 Ion Formation from Inorganic Samples 483

10.2.2 Ion Formation from Organic Samples 484

10.3 Liquid Matrices for FAB and LSIMS 486

10.3.1 The Role of the Liquid Matrix 486

10.3.2 FAB Matrix Spectra – General Characteristics 487

10.3.3 Unwanted Reactions in FAB-MS 487

10.4 Applications of FAB-MS 488

10.4.1 FAB-MS of Analytes of Low to Medium Polarity 488

10.4.2 FAB-MS of Ionic Analytes 490

10.4.3 High-Mass Analytes in FAB-MS 491

10.4.4 Accurate Mass Measurements in FAB Mode 492

10.4.5 Continuous-Flow FAB 494

10.4.6 Low-Temperature FAB 495

10.4.7 FAB-MS and Peptide Sequencing 496

10.5 FAB and LSIMS – General Characteristics 496

10.5.1 Sensitivity of FAB-MS 496

10.5.2 Types of Ions in FAB-MS 497

10.5.3 Analytes for FAB-MS 497

10.5.4 Mass Analyzers for FAB-MS 497

10.6 Massive Cluster Impact 498

10.7 252Californium Plasma Desorption 498

References 499

11 Matrix-Assisted Laser Desorption/Ionization 507

Learning Objectives 50711.1 Ion Sources for LDI and MALDI 50811.2 Ion Formation 50911.2.1 Ion Yield and Laser Fluence 51011.2.2 Effect of Laser Irradiation on the Surface 51111.2.3 Temporal Evolution of a Laser Desorption Plume 51211.2.4 Processes of Ion Formation in MALDI 51311.2.5 “Lucky Survivor” Model of Ion Formation 51411.3 MALDI Matrices 51611.3.1 Role of the Solid Matrix 51611.3.2 Matrices in UV-MALDI 51611.3.3 Characteristics of MALDI Matrix Spectra 51911.4 Sample Preparation 51911.4.1 MALDI Target 51911.4.2 Standard Sample Preparation 52011.4.3 Cationization 52211.4.4 Cation Removal 52411.4.5 Solvent-Free Sample Preparation 52611.4.6 Additional Methods of Sample Supply 52711.5 Applications of LDI 52711.6 Applications of MALDI 52911.6.1 Protein Analysis by MALDI-MS 52911.6.2 Peptide Sequencing and Proteomics 53111.6.3 Carbohydrate Analysis by MALDI-MS 53611.6.4 Oligonucleotide Analysis by MALDI-MS 53811.6.5 MALDI-MS of Synthetic Polymers 53911.7 Special Surfaces to Mimic the Matrix 54111.7.1 Desorption/Ionization on Silicon 54111.7.2 Nano-Assisted Laser Desorption/Ionization 54211.7.3 Further Variations of the MALDI Theme 54311.8 MALDI Imaging 54411.9 Atmospheric Pressure MALDI 54611.10 General Characteristics of MALDI 54711.10.1 Sample Consumption and Detection Limit 54711.10.2 Analytes for MALDI 54711.10.3 Types of Ions in LDI and MALDI-MS 54811.10.4 Mass Analyzers for MALDI-MS 548References 549

12 Electrospray Ionization 561

Learning Objectives 56112.1 Development of ESI and Related Methods 56212.1.1 Atmospheric Pressure Ionization 56312.1.2 Thermospray 56412.1.3 Electrohydrodynamic Ionization 565

12.3.3 Nanoelectrospray from a Chip 57712.4 Ion Formation in ESI 57812.4.1 Formation of the Electrospray Plume 57812.4.2 Disintegration of Charged Droplets 58112.4.3 Formation of Ions from Charged Droplets 58212.5 Multiply Charged Ions and Charge Deconvolution 58512.5.1 Dealing with Multiply Charged Ions 58512.5.2 Mathematical Charge Deconvolution 58712.5.3 Computerized Charge Deconvolution 58812.5.4 Hardware Charge Deconvolution 59012.5.5 Controlled Charge Reduction in ESI 59212.6 Applications of ESI-MS 59312.6.1 ESI-MS of Small Molecules 59312.6.2 ESI of Metal Complexes 59412.6.3 ESI of Surfactants 59612.6.4 Oligonucleotides, DNA, and RNA 59612.6.5 ESI-MS of Oligosaccharides 59912.6.6 High-Mass Proteins and Protein Complexes 60012.7 Summary of ESI Characteristics 60112.7.1 Sample Consumption 60312.7.2 Types of Ions in ESI 60312.7.3 Mass Analyzers for ESI 60312.8 Atmospheric Pressure Chemical Ionization 60412.8.1 Ion Sources for APCI 60412.8.2 Ion Formation in APCI 60512.8.3 APCI Spectra 60512.9 Atmospheric Pressure Photoionization 60812.9.1 Ion Formation in APPI 60812.9.2 APPI Spectra 610References 612

13 Ambient Mass Spectrometry 621

Learning Objectives 62113.1 Desorption Electrospray Ionization 62213.1.1 Experimental Setup for DESI 62213.1.2 Mechanisms of Ion Formation in DESI 626

13.1.3 Analytical Features of DESI 62713.2 Desorption Atmospheric Pressure Chemical Ionization 63113.3 Desorption Atmospheric Pressure Photoionization 63213.4 Other Methods Related to DESI 63413.4.1 Desorption Sonic Spray Ionization 63513.4.2 Extractive Electrospray Ionization 63513.4.3 Electrospray-Assisted Laser Desorption/Ionization (ELDI) 63713.4.4 Laser Ablation Electrospray Ionization 63813.4.5 Atmospheric Pressure Solids Analysis Probe 64013.5 Direct Analysis in Real Time 64013.5.1 Experimental Setup for DART 64013.5.2 Ion Formation in DART 64213.5.3 Analytical Applications of DART 64213.6 Overview of Ambient Mass Spectrometry 644References 645

14 Hyphenated Methods 651

Learning Objectives 65114.1 Concept of Chromatography-Mass Spectrometry 65214.1.1 Ion Chromatograms 65314.1.2 Repetitive Acquisition of Mass Spectra During Elution 65414.1.3 Selected Ion Monitoring 65614.1.4 Selected Reaction Monitoring 65814.2 Quantitation 65914.2.1 Quantitation by External Standardization 65914.2.2 Quantitation by Internal Standardization 66014.2.3 Quantitation by Isotope Dilution 66114.2.4 Retention Times of Isotopologs 66314.3 Gas Chromatography-Mass Spectrometry 66314.3.1 GC-MS Interfaces 66314.3.2 Volatility and Derivatization 66414.3.3 Column Bleed 66514.3.4 Fast GC-MS 66714.3.5 Multiplexing for Increased Throughput 66714.4 Liquid Chromatography-Mass Spectrometry 66814.4.1 Multiplexed LC-ESI-MS 67114.5 Ion Mobility Spectrometry-Mass Spectrometry 67314.6 Tandem MS as a Complement to LC-MS 67514.7 Ultrahigh-Resolution Mass Spectrometry 678References 680

15 Inorganic Mass Spectrometry 685

Learning Objectives 68515.1 Thermal Ionization Mass Spectrometry 68915.2 Spark Source Mass Spectrometry 69115.3 Glow Discharge Mass Spectrometry 694

Appendix 717

A.1 Units, Physical Quantities, and Physical Constants 717A.2 Isotopic Composition of the Elements 718A.3 Carbon Isotopic Patterns 725A.4 Chlorine and Bromine Isotopic Patterns 726A.5 Silicon and Sulfur Isotopic Patterns 727A.6 Isotopologs and Accurate Mass 727A.7 Characteristic Ions 728A.8 Common Impurities 729A.9 Amino Acids 730A.10 Method Selection Guide 731A.11 How to Recognize Cationization 732A.12 Systematic Approach to Mass Spectra 733A.13 Rules for the Interpretation of Mass Spectra 733A.14 Nobel Prizes for Mass Spectrometry 734

Subject Index 735

• How mass spectra are displayed and communicated

• The performance features of mass spectrometry

• Basic terminology and conventions in data presentation

Mass spectrometry is an indispensable analytical tool in chemistry, biochemistry, pharmacy, medicine, and many related fields of science No student, researcher or practitioner in these disciplines can really get by without a substantial knowledge

of mass spectrometry Can this statement be approved?

Mass spectrometry (MS) is employed to analyze combinatorial libraries [1,2] sequence biomolecules [3], and help explore single cells [4,5] or objects from outer space [6] Structure elucidation of unknown substances, environmental and forensic analytes, quality control of drugs, foods, and polymers: they all rely to a great extent on mass spectrometry [7-13] Today, “mass spectrometry is inter-woven with biology to an extent that basic considerations of proteomics research are dealt with in a MS journal” [14]

Whatever the analytical interest may be: mass spectrometry aims to identify a compound from the molecular or atomic mass(es) of its constituents The informa-tion delivered by mass alone can be sufficient for the identification of elements and the determination of the molecular formula of an analyte The relative abun-dance of isotopologs helps to decide which elements contribute to such a formula and to estimate the number of atoms of a contributing element Under the condi-tions of certain mass spectrometric experiments, fragmentation of ions can deliver information on ionic structure Thus, MS elucidates the connectivity of atoms within smaller molecules, identifies functional groups, determines the (average) number and eventually the sequence of constituents of macromolecules, and in some cases even yields their three-dimensional structure From the 1950s to the present, mass spectrometry has developed in big strides and innovations are still being made at an enormous rate [15,16] The pioneering mass spectrometrist worked with home-built rather than commercial instruments These machines, typically magnetic sector instruments using electron ionization, delivered a few mass spectra per day, providing that the device was delicately handled Intimate knowledge of such an instrument and interpretation skills of the according EI spectra would provide the mass spectrometrist with a previously unknown wealth

J Gross, Mass Spectrometry

© Springer-Verlag Berlin Heidelberg 2011

, 2nd ed., DOI 10.1007/978-3-642-10711-5_1,

of insight into structural details [17-22] In particular the life sciences gave a great impetus for new developments for expanding the mass range to higher molecular weights and increasingly fragile molecules

Table 1.1 Fields of application of mass spectrometry

Key application and field of application Explanation

Elemental and isotopic analysis

abun-Organic and bio-organic analysis

Organic chemistry

Polymer chemistry

Biochemistry and medicine

Identification and structural characterization

of molecules from small to very large as vided either by chemistry, physiological processes, or polymer chemistry

pro-Structure elucidation

Organic chemistry

Polymer chemistry

Biochemistry and medicine

Mass spectrometric experiments can be ranged consecutively to study mass-selected ions in tandem mass spectrometry (MS/MS or

ar-MS 2 ) Eventually products are subjected to a third level (MS 3 ) and so forth (MS n )

Characterization of ionic species

and chemical reactions

Physical chemistry

Thermochemistry

Tandem MS provides an elegant means for the study of unimolecular or bimolecular re- actions of gas phase ions and for the determi- nation of ion energetics

Coupling to separation techniques

Mass spectral imaging

Biomedical studies

Material sciences

Mass spectra can be obtained from ter-sized areas on surfaces, translating the lat- eral distribution of compounds on surfaces (microelectronics, slices of tissue) into im- ages, which in turn can be correlated to opti- cal images

Port-by the mere task of selecting one out of about a dozen of promising techniques available for their particular sample It is precisely this diversity that makes a basic understanding of the concepts and tools of mass spectrometry more important than ever On the other extreme, there are mass spectrometry laboratories specialized

on employing only one particular method – preferably matrix-assisted laser sorption/ionization (MALDI) or electrospray ionization (ESI) In contrast to some

de-50 years ago, the instrumentation is now concealed in a sort of “black box”, more appealingly designed to resemble an espresso machine or tumble dryer Let us take a look inside!

1.1 Aims and Scope

This book is tailored to be your guide to mass spectrometry – from the first steps

to your daily work in research Starting from the very principles of gas phase ion chemistry and isotopic properties, it leads you through the design of mass analyz-ers, mass spectral interpretation, and applied ionization methods The book closes with chapters on chromatography–mass spectrometry coupling and one on inor-ganic mass spectrometry In total, it comprises fifteen chapters that can be read in-dependently from each other However, for the novice it is recommended to work through from start to finish, occasionally skipping over more advanced sections (Table 1.1) Now in its 2nd edition, “Mass Spectrometry – A Textbook” continues

to be your companion from undergraduate to graduate studies in chemistry, chemistry, and other natural sciences, and aims to hold its value when serving as a hands-on reference in the course of professional life

bio-Step by step you will understand how mass spectrometry works and what it can

do as a powerful tool in your hands – equally well for analytical applications as for basic research A clear layout and many high-quality figures will make is eas-ier to acquire the new knowledge Many tables have been included, compiling facts and comparing topics Interrelationships are pointed to where appropriate The correctness of scientific content has been examined by leading experts Each chapter begins with a set of Learning Objectives and closes by providing an elabo-rate list of references, emphasizing tutorial and review articles, book chapters, and monographs in the respective field Titles are included with all citations to help

with the evaluation of useful further reading [23] References for general further reading on mass spectrometry are compiled at the end of this Introduction

The coverage of this book is basically restricted to what is called “organic mass spectrometry” in a broad sense It includes the ionization methods and mass ana-lyzers currently in use, and in addition to classical organic compounds it covers applications to bio-organic samples such as peptides and oligonucleotides Of course, transition metal complexes, synthetic polymers, and fullerenes are dis-cussed as well as environmental or forensic applications Elemental analysis, the classical field of inorganic mass spectrometry has been added to get a taste of mass spectrometry beyond molecular species

Table 1.2 Chapters of this book: overview for orientation

2 Principles of Ionization and Ion Dissociation

3 Isotopic Composition and Accurate Mass

4 Instrumentation

Tools of the trade Basics needed for the understanding of any of the subsequent chapters

5 Practical Aspects of Electron Ionization

6 Fragmentation of Organic Ions and

Interpretation of EI Mass Spectra

Electron ionization: the cal key to organic MS and in- dispensable part of every intro- ductory course

classi-7 Chemical Ionization

8 Field Ionization and Field Desorption

Traditional, nonetheless still highly relevant soft ionization methods

9 Tandem Mass Spectrometry Fully controlled dissociation of

mass-selected ions for many teresting purposes

in-10 Fast Atom Bombardment

11 Matrix-Assisted Laser Desorption/Ionization

12 Electropray Ionization

More soft ionization methods The latter two represent today's most relevant techniques in MS

13 Ambient Mass Spectrometry Exciting new field based on

ad-vances in atmospheric pressure ionization methods

tech-niques to MS

15 Inorganic Mass Spectrometry There is even more: a glimpse

beyond the horizon of organic and biomedical MS

redording and presentation of mass spectra – and what’s more is the art of preting mass spectra All these aspects are correlated to each other in many ways and in their entirety contribute to what is referred to as mass spectrometry (Fig 1.1) In other words, mass spectrometry is multifacet rather than to be viewed from a single perspective Like a view onto a globe does not reveal the complete surface of our planet, but roughly just one continent at a time, mass spectrometry needs to be explored from various vantage points [24]

coupling of separation devices

interpretation of mass spectra

fragmentation pathways characteristic ions rules for interpretation

1.2 What Is Mass Spectrometry?

Now, what is mass spectrometry? Well in any case, mass spectrometry is special

in many ways Up front, most mass spectrometrists do not fathom to be addressed

as mass spectroscopists

Hint: “First of all, never make the mistake of calling it 'mass spectroscopy'

Spectroscopy involves the absorption of electromagnetic radiation, and mass spectrometry is different, as we will see The mass spectrometrists sometimes get upset if you confuse this issue” [25]

Indeed, there is almost no book using the term mass spectroscopy and all tific journals in the field bear mass spectrometry in their titles You will find such

scien-highlighted rules, hints, notes, and definitions throughout the book This more amusing one – we might call it the “zeroth law of mass spectrometry” – has been taken from a standard organic chemistry textbook The same author completes his chapter on mass spectrometry with the conclusion that “despite occasional myster-ies, mass spectrometry is still highly useful” [25]

Historical Remark: Another explanation for this terminology originates from

the historical development of our instrumentation [16] The device employed

by Thomson for the first mass-separating experiments was a type of

spectro-scope showing blurred signals on a fluorescent screen [26] Dempster

con-structed an instrument with a deflecting magnetic field angled at 180° In order

to detect different masses, it could either be equipped with a photographic plate

– a so-called mass spectrograph – or it could have a variable magnetic field to

detect different masses by focusing them successively onto an electric point

de-tector [27] Later, the term mass spectrometer was coined for the latter type of instruments using a scanning magnetic field [28]

1.2.1 Mass Spectrometry

“The basic principle of mass spectrometry (MS) is to generate ions from either

in-organic or in-organic compounds by any suitable method, to separate these ions by

their mass-to-charge ratio (m/z) and to detect them qualitatively and quantitatively

by their respective m/z and abundance The analyte may be ionized thermally, by

electric fields or by impacting energetic electrons, ions or photons The ions can

be single ionized atoms, clusters, molecules or their fragments or associates Ion separation is effected by static or dynamic electric or magnetic fields.” Although this definition of mass spectrometry dates back to 1968 when organic mass spec-trometry was in its infancy [29], it is still valid However, some additions should

be made First, ionization of a sample can be effected not only by electrons, but also by (atomic) ions or photons, energetic neutral atoms, electronically excited atoms, massive cluster ions, and even electrostatically charged microdroplets can also be used to effect Second, as demonstrated with great success by the time-of-

flight analyzer, ion separation by m/z can also be effected in field-free regions,

provided the ions possess a well-defined kinetic energy at the entrance of the flight path

Fig 1.2 Mass spectrometric techniques for different needs arranged by main fields of

ap-plication and estimated relative hardness or softness Reproduced from Ref [24] by sion © Wiley-VCH, Weinheim, 2009

permis-1.2.2 Mass Spectrometer

Obviously, almost any technique to achieve the goals of ionization, separation and detection of ions in the gas phase can be applied – and actually has been applied –

in mass spectrometry Fortunately, there is a simple basic scheme that all mass

spectrometers follow A mass spectrometer consists of an ion source, a mass

ana-lyzer, and a detector which are operated under high vacuum conditions A closer

look at the front end of such a device might separate the steps of sample

introduc-tion, evaporaintroduc-tion, and successive ionization or desorption/ionizaintroduc-tion, respectively,

but it is not always trivial to identify each of these steps as clearly separated from each other If the manufacturing date of the instrument is relatively recent, it will have a data system which collects and processes data from the detector Since the 1990s, mass spectrometers are fully equipped and controlled by data systems (Fig 1.3)

The consumption of analyte by its examination in the mass spectrometer is an

aspect deserving our attention: Whereas other spectroscopic methods such as clear magnetic resonance (NMR), infrared (IR) or Raman spectroscopy do allow for sample recovery, mass spectrometry is destructive, i.e., it consumes the ana-lyte This is apparent from the process of ionization and translational motion through the mass analyzer to the detector during analysis Although some sample

nu-is consumed, it may still be regarded as practically nondestructive, however, cause the amount of analyte needed is in the low microgram range or even by sev-eral orders of magnitude below In turn, the extremely low sample consumption of mass spectrometry makes it the method of choice when most other analytical techniques fail because they are not able to yield analytical information from nanogram amounts of sample

be-ionsource analyzermass detector systemdata

sample

inlet

atmosphere/

Fig 1.3 General scheme of any mass spectrometer Often, several types of sample inlets

are attached to the ion source housing Transfer of the sample from atmospheric pressure to the high vacuum of the ion source and mass analyzer is accomplished by use of a vacuum lock (Chap 5.2)

1.2.3 Mass Scale

Plotting mass spectra on a physical scale of mass per electric charge (kg C–1) would be very inconvenient to use Thus, mass spectrometrists have adopted the use of a scale of atomic mass per number of elementary charges and termed it

mass-to-charge ratio, m/z, (read “m over z” and write m/z) [30] There is only one

correct writing convention: the location of a peak on the abscissa is to be reported

as “at m/z x”

Unfortunately, m/z is a rather artificial construct as it has not received the status

of a physical unit Instead, m/z is dimensionless by definition It may be

under-stood as the ratio of the numerical value of ionic mass on the atomic mass scale and the number of elementary charges of the respective ion The number of ele-mentary charges is often, but by far not necessarily, equal to one As long as only

singly charged ions are observed (z = 1) the m/z scale directly reflects the atomic

mass scale However, there can be conditions where doubly, triply, or even highly charged ions are being created from the analyte depending on the ionization me-thod employed

cause the mass spectrometer detects only charged species, i.e., the

charge-retaining group of a fragmenting ion Since 1961 the unified atomic mass [u] has

been defined as 1/12 of the mass of one single atom of the nuclide 12C which by convention has been set to precisely 12 u (Chap 3)

Note: Mass spectrometrists working in the biomedical field tend to use the

dal-ton [Da] (to honor J Daldal-ton) instead of the unified atomic mass [u] The daldal-ton

also is not an SI unit The dalton is equivalent to unified atomic mass in that there is no conversion factor between these units

1.2.4 Mass Spectrum

A mass spectrum is the two-dimensional representation of signal intensity nate) versus m/z (abscissa) The position of a peak, as signals are usually called, reflects the m/z of an ion that has been created from the analyte within the ion source The intensity of this peak correlates to the abundance of that ion

(ordi-Often but not necessarily, the peak at highest m/z results from the detection of the intact ionized molecule, the molecular ion, M+• The molecular ion peak is usually accompanied by several peaks at lower m/z caused by fragmentation of the molecular ion to yield fragment ions Consequently, the respective peaks in the mass spectrum may be referred to as fragment ion peaks

The most intense peak of a mass spectrum is called base peak In most

repre-sentations of mass spectral data the intensity of the base peak is normalized to

100% relative intensity This largely helps to make mass spectra more easily

com-parable The normalization can be done because the relative intensities are cally independent from the absolute ion abundances registered by the detector

basi-Note: There is an upper limit for the number of ions and neutrals per volume

inside the ion source where the appearance of spectra will significantly change due to ion–molecule reactions One should also be aware of the fact that a sin-

gle molecule only yields one ion of one m/z value To build a useful mass

spec-trum requires the statistics of thousands of ion formations and eventual

frag-mentations to result in signals at different m/z where each of them can be

assigned a relevant intensity

Example: In the electron ionization mass spectrum of a particular hydrocarbon,

the molecular ion peak and the base peak of the spectrum happen to correspond to

the same ionic species at m/z 16 (Fig 1.4) The fragment ion peaks at m/z 12–15 are spaced at Δ(m/z) = 1 Obviously, the molecular ion, M+•, fragments by loss of

H• which is the only possibility to explain the peak at m/z 15 by loss of a neutral of

1 u mass Accordingly, the peaks at lower m/z might arise from loss of an H2

molecule (2 u) and so forth It is rather obvious that this spectrum corresponds to methane, CH4, showing its molecular ion peak at m/z 16 because the atomic mass

of carbon is 12 u and that of hydrogen is 1 u, and therefore 12 u + 4 × 1 u = 16 u Removal of one electron from a 16 u neutral yields a singly-charged radical ion

that is detected at m/z 16 by the mass spectrometer Of course, most mass spectra

are not that simple, but this is how it works

Fig 1.4 Electron ionization mass spectrum of a hydrocarbon Adapted with permission

© National Institute of Standards and Technology, NIST, 2002

The above spectrum is represented as a bar graph or histogram Such data

duction is common in mass spectrometry and useful as long as peaks are well

re-solved The intensities of the peaks can be obtained either from measured peak

heights or more correctly from peak areas The position, i.e., the m/z ratio, of the

signal is determined from its centroid Noise below some user-defined cut level is usually subtracted from the bar graph spectrum If peak shape and peak width be-come important, e.g., in case of high-mass analytes or high-resolution measure-

ments, spectra should be represented as profile data as initially acquired by the mass spectrometer Tabular listings of mass spectra are used to report mass and

intensity data more accurately (Fig 1.5)

Fig 1.5 Tetrapentacontane, C54H 110 : Three representations of the molecular ion signal in

the field desorption mass spectrum (Chap 8) (a) profile spectrum, (b) bar graph tion, and (c) tabular listing

representa-1.3 Ion Chromatograms

In gas and liquid chromatographs the components of a mixture are eluted at

differ-ent retdiffer-ention times from the chromatographic column When a mass spectrometer

is employed as the chromatographic detector (GC-MS and LC-MS, Chaps 5.4, 5.5 and 14) its output must somehow represent the chromatogram that would have been obtained with other chromatographic detectors (FID, TCD, UV) The chro-

matogram as produced by the mass spectrometer is composed of a large set of

consecutively acquired mass spectra, each of which containing spectral data of the

eluting species, i.e., each component can be identified from its mass spectrum Because mass spectral chromatograms represent ionic abundances as a function of

retention time, these are termed ion chromatograms To a certain degree

fractiona-tion is also observed during evaporafractiona-tion of mixtures from a direct inserfractiona-tion probe, though the separation by far can not be compared to that of chromatographic sys-tems

Note: The terms ion profile and ion pherogram have been suggested in place of

ion chromatogram when DIPs or electrophoresis are used, respectively, as

these cannot produce chromatograms in the strict sense [31,32] However, this would add yet more terms to one of the rather vague areas of our terminology which is even insufficiently covered by official recommendations [31-36]

The total ion current (TIC) can either be measured by a hardware TIC monitor before mass analysis (nA to µA range), or its equivalent can be reconstructed or

extracted after mass analysis [33] Both adjectives, reconstructed and extracted,

serve to illustrate that the chromatogram has been obtained from a set of spectra

by a computational process that selects user-defined signals to build the trace Thus, the TIC represents a measure of the overall intensity of ion production or

of mass spectral output as a function of time, respectively The TIC obtained by

means of data reduction [34], i.e., by summation of peak intensities of each mass spectrum as successively acquired during analysis, is termed total ion chroma-

togram (TIC) For this purpose, the sum of all ion intensities belonging to each of

the spectra is plotted as a function of time or scan number, respectively The term

total ion current chromatogram (TICC) refers to a chromatogram obtained by

plotting the total ion current detected in each of a series of mass spectra recorded

as a function of retention times of the chromatographically separated components

of a mixture (which essentially is implicated by: TIC) Sometimes we find

combi-nations such as reconstructed total ion current (RTIC) or reconstructed total ion

current chromatogram (RTICC)

Note: Modern instruments do not support hardware TIC measurements, but

un-til the 1970s, there used to be a hardware TIC monitor on the electronics panel The TIC was obtained by measuring the ion current caused by those ions hitting the ion source exit plate instead of passing through its slit

The term reconstructed ion chromatogram (RIC) was and still is used by many

to describe the intensity of a given m/z or m/z range plotted as a function of time

or scan number Recently, the term extracted ion chromatogram (EIC) has been

used to describe a chromatogram created by plotting the intensity of the signal

ob-served at a chosen m/z value or set of values in a series of mass spectra recorded

as a function of retention time Plotting RICs or EICs is especially useful to

iden-tify a target compound of known m/z from complex GC-MS or LC-MS data In

other words, the RIC allows to retrieve the retention time of the target compound

RICs can also be used to uncover the relationship of certain m/z values to different

mass spectra obtained from the measurement of a single (impure) sample Thus, RICs (EICs) often reveal valuable information on impurities accompanying the main product, e.g., remaining solvents, plasticizers, vacuum grease, or synthetic by-products (Chap 5.2.4)

Finally, the base peak chromatogram (BPC) is a chromatogram obtained by

plotting the signal of the ions giving the base peak detected in each of a series of mass spectra recorded as a function of retention time

Example: Polycyclic and nitro musks are frequently used as fragrances in

cos-metic products A GC-MC procedure for their identification and quantitation ploys the characteristic RICs (EICs) of tonalide (AHTN), C18H26O+•, m/z 258, and

em-xylene musk (MX), C12H15N3O6+•, m/z 297 [37] Although eluting simultaneously

(peak 3 in Fig 1.6), EICs allow to separate both components by choosing

charac-teristic m/z values, e.g., of the respective molecular ions or abundant fragment

ions The concentration of the solution injected was in the order of 1 µg ml–1 per component

Fig 1.6 Typical GC-MS chromatogram, i.e., TIC (TICC) in full scan mode for synthetic

musks and some standards The inset shows the characteristic RICs (EICs) of tonalide (AHTN), C18H26O +•, m/z 258, and xylene musk (MX), C12H15N3O6+•, m/z 297 Reproduced

from Ref [37] with permission © The Japan Society for Analytical Chemistry, 2009

1.4 Performance of Mass Spectrometers

spec-1 for solids [33]; for gaseous analytes, it can be specified as the ratio of ion current

to analyte partial pressure in units of A Pa–1 [31,34]

According to the above definition, sensitivity does not only depend on the zation efficiency of EI or any other ionization method Also relevant are the ex-traction of ions from the ion source, the mass range acquired during the experi-ment, and the transmission of the mass analyzer Therefore, the complete

ioni-experimental conditions have to be stated with sensitivity data

Example: Modern magnetic sector instruments are specified to have a

sensitiv-ity of about 4 × 10–7 C µg–1 for the molecular ion of methylstearate, m/z 298, at

R = 1000 in 70 eV EI mode The charge of 4 × 10–7 C corresponds to 2.5 × 1012

electron charges One microgram of methylstearate is equivalent to 3.4 × 10–9 mol

or 2.0 × 1015 molecules

1.4.2 Detection Limit

The term detection limit or limit of detection (LOD) is almost self-explanatory, yet

it is often confused with sensitivity The detection limit defines the smallest flow

or the lowest amount of analyte necessary to obtain a signal that can be guished from the background noise The detection limit is valid for one well-specified analyte upon treatment according to a certain analytical protocol [31,33,34]

distin-Of course, the sensitivity of an instrumental setup is of key importance to low detection limits; nevertheless, the detection limit is a clearly different quantity The detection limit may either be stated as a relative measure in trace analysis, e.g., 1 ppb of dioxin in waste oil samples (equivalent to 1 µg kg–1 of sample), or as

an absolute measure, e.g., 1 femtomol of substance P in MALDI-MS

1.4.3 Signal-to-Noise Ratio

The signal-to-noise ratio (S/N) describes the uncertainty of an intensity

measure-ment and provides a quantitative measure of a signal's quality by quantifying the

ratio of the intensity of a signal relative to noise

Noise results from the electronics of an instrument, and thus noise is not only

present between the signals but also on the signals Consequently, intensity

meas-urements are influenced by noise Real and very numerous background signals of various origin, e.g., FAB or MALDI matrices, GC column bleed and impurities, can appear as if they were electronic noise, so-called “chemical” noise In the strict sense, this should be distinct from electronic noise and should be reported as

signal-to-background ratio (S/B) [31] In practice, this can be difficult to do

Noise is statistical in nature, and therefore can be reduced by elongated data acquisition and subsequent summing or averaging of the spectra, respectively Ac-cordingly, an intensive peak has a better S/N than a low-intensity peak within the same spectrum The reduction of noise is proportional to the square root of acqui-sition time or number of single spectra that are averaged [38], e.g., the noise is re-duced by a factor of 3.16 by averaging 10 spectra or by a factor of 10 by averaging

100 spectra, respectively

Example: A signal may be regarded to be clearly visible at S/N ≥ 10, a value

often stated with detection limits A mass spectrometer in good condition yields S/N > 104 which means in turn that even isotopic peaks of low relative intensity can be reliably measured, provided there is no interference with background sig-nals (Fig 1.7)