Part one sets the stage with a general description of NMR introducing important parameters such as the chemical shift and scalar or dipolar couplings. Part two describes the theory behind NMR, providing a profound understanding of the involved spin physics, deliberately kept shorter than in other NMR textbooks, and without a rigorous mathematical treatment of all the physico-chemical computations. Part three discusses technical and practical aspects of how to use NMR. Important phenomena such as relaxation, exchange, or the nuclear Overhauser effects and the methods of modern NMR spectroscopy including multidimensional experiments, solid state NMR, and the measurement of molecular interactions are the subject of part four. The final part explains the use of NMR for the structure determination of selected classes of complex biomolecules, from steroids to peptides or proteins, nucleic acids, and carbohydrates.
For chemists as well as users of NMR technology in the biological sciences.
Oliver Zerbe is the head of the NMR department at the University of Zurich. He studied chemistry and obtained his PhD under the supervision of Wolfgang von Philipsborn in Zurich. After a postdoctoral stay in the group of Kurt Wuthrich at the ETH Zurich he conducted his habilitation with Gerd Folkers at the Institute of Pharmaceutical Sciences at the ETH. In 2003 he returned to his present location at the University of Zurich, where he is now a professor in the Department of Chemistry. His main interests are structures of proteins, particularly of membrane proteins. Oliver Zerbe is the author of approximately 100 scientific publications in peer-reviewed journals and has edited one book, 'NMR in drug research'. After studying chemistry at the University of Applied Sciences of Bern,
Simon Jurt has been working for more than ten years in the NMR department of the University of Zurich. In addition to maintenance and trouble shooting of the NMR spectrometers, he introduces the students to the secrets of NMR spectroscopy, teaches practical NMR courses and is involved in several research projects. His main interests are the experimental NMR techniques, which allow obtaining a plethora of chemo-physical information from the spin physics.
Oliver Zerbe is the head of the NMR department at the University of Zurich. He studied chemistry and obtained his PhD under the supervision of Wolfgang von Philipsborn in Zurich. After a postdoctoral stay in the group of Kurt Wüthrich at the ETH Zurich he conducted his habilitation with Gerd Folkers at the Institute of Pharmaceutical Sciences at the ETH. In 2003 he returned to his present location at the University of Zurich, where he is now a professor in the Department of Chemistry. His main interests are structures of proteins, particularly of membrane proteins. Oliver Zerbe is the author of approximately 100 scientific publications in peer-reviewed journals and has edited one book, "NMR in drug research". After studying chemistry at the University of Applied Sciences of Bern, Simon Jurt has been working for more than ten years in the NMR department of the University of Zurich. In addition to maintenance and trouble shooting of the NMR spectrometers, he introduces the students to the secrets of NMR spectroscopy, teaches practical NMR courses and is involved in several research projects. His main interests are the experimental NMR techniques, which allow obtaining a plethora of chemo-physical information from the spin physics.
Applied NMR Spectroscopy for Chemists and Life Scientists 1
Contents 7
Preface 17
1 Introduction to NMR Spectroscopy 21
1.1 Our First 1D Spectrum 21
1.2 Some Nomenclature: Chemical Shifts, Line Widths, and Scalar Couplings 22
1.3 Interpretation of Spectra: A Simple Example 25
1.4 Two-Dimensional NMR Spectroscopy: An Introduction 29
Part One Basics of Solution NMR 31
2 Basics of 1D NMR Spectroscopy 33
2.1 The Principles of NMR Spectroscopy 33
2.2 The Chemical Shift 36
2.3 Scalar Couplings 37
2.4 Relaxation and the Nuclear Overhauser Effect 40
2.5 Practical Aspects 43
2.5.1 Sample Preparation 43
2.5.2 Referencing 45
2.5.3 Sensitivity and Accumulation of Spectra 47
2.5.4 Temperature Calibration 49
2.6 Problems 50
Further Reading 51
3 1H NMR 53
3.1 General Aspects 53
3.2 Chemical Shifts 54
3.2.1 Influence of Electronegativity of Substituents 55
3.2.2 Anisotropy Effects 55
3.2.3 Other Factors Affecting Chemical Shifts: Solvent, Temperature, pH, and Hydrogen Bonding 57
3.2.4 Shift Reagents 57
3.3 Spin Systems, Symmetry, and Chemical or Magnetic Equivalence 59
3.3.1 Homotopic, Enantiotopic, and Diastereotopic Protons 62
3.3.2 Determination of Enantiomeric Purity 63
3.4 Scalar Coupling 64
3.4.1 First-Order Spectra 65
3.4.2 Higher-Order Spectra and Chemical Shift Separation 67
3.4.3 Higher-Order Spectra and Magnetic Equivalence 69
3.5 1H–1H Coupling Constants 70
3.5.1 Geminal Couplings 70
3.5.2 Vicinal Couplings 70
3.5.3 Long-Range Couplings 72
3.5.4 1H Couplings to Other Nuclei 72
3.6 Problems 74
Further Reading 75
4 NMR of 13C and Heteronuclei 77
4.1 Properties of Heteronuclei 77
4.2 Indirect Detection of Spin-1/2 Nuclei 79
4.3 13C NMR Spectroscopy 79
4.3.1 The 13C Chemical Shift 80
4.3.2 X,13C Scalar Couplings 84
4.3.3 Longitudinal Relaxation of 13C Nuclei 88
4.3.4 Recording 13C NMR Spectra 88
4.4 NMR of Other Main Group Elements 90
4.4.1 Main Group Nuclei with I=1/2 91
4.4.2 Main Group Nuclei with I> 1/2
4.5 NMR Experiments with Transition Metal Nuclei 98
4.5.1 Technical Aspects of Inverse Experiments with I=1/2 Metal Nuclei 99
4.5.2 Experiments with Spin I> 1/2 Transition Metal Nuclei
4.6 Problems 102
Further Reading 104
Part Two Theory of NMR Spectroscopy 105
5 Nuclear Magnetism – A Microscopic View 107
5.1 The Origin of Magnetism 107
5.2 Spin – An Intrinsic Property of Many Particles 108
5.3 Experimental Evidence for the Quantization of the Dipole Moment: The Stern–Gerlach Experiment 113
5.4 The Nuclear Spin and Its Magnetic Dipole Moment 114
5.5 Nuclear Dipole Moments in a Homogeneous Magnetic Field: The Zeeman Effect 116
5.5.1 Spin Precession 118
5.6 Problems 123
6 Magnetization – A Macroscopic View 125
6.1 The Macroscopic Magnetization 125
6.2 Magnetization at Thermal Equilibrium 126
6.3 Transverse Magnetization and Coherences 128
6.4 Time Evolution of Magnetization 129
6.4.1 The Bloch Equations 130
6.4.2 Longitudinal and Transverse Relaxation 132
6.5 The Rotating Frame of Reference 135
6.6 RF Pulses 137
6.6.1 Decomposition of the RF Field 138
6.6.2 Magnetic Fields in the Rotating Frame 139
6.6.3 The Bloch Equations in the Rotating Frame 140
6.6.4 Rotation of On-Resonant and Off-Resonant Magnetizationunder the Influence of Pulses 141
6.7 Problems 142
7 Chemical Shift and Scalar and Dipolar Couplings 145
7.1 Chemical Shielding 145
7.1.1 The Contributions to Shielding 147
7.1.2 The Chemical Shifts of Paramagnetic Compounds 151
7.1.3 The Shielding Tensor 152
7.2 The Spin–Spin Coupling 153
7.2.1 Scalar Coupling 154
7.2.2 Quadrupolar Coupling 160
7.2.3 Dipolar Coupling 161
7.3 Problems 164
Further Reading 165
8 A Formal Description of NMR Experiments: The Product Operator Formalism 167
8.1 Description of Events by Product Operators 168
8.2 Classification of Spin Terms Used in the POF 169
8.3 Coherence Transfer Steps 171
8.4 An Example Calculation for a Simple 1D Experiment 172
Further Reading 173
9 A Brief Introduction into the Quantum-Mechanical Concepts of NMR 175
9.1 Wave Functions, Operators, and Probabilities 175
9.1.1 Eigenstates and Superposition States 176
9.1.2 Observables of Quantum-Mechanical Systemsand Their Measured Quantities 177
9.2 Mathematical Tools in the Quantum Description of NMR 178
9.2.1 Vector Spaces, Bra's, Ket's, and Matrices 178
9.2.2 Dirac's Bra–Ket Notation 179
9.2.3 Matrix Representation of State Vectors 180
9.2.4 Rotations between State Vectors can be Accomplished with Tensors 181
9.2.5 Projection Operators 182
9.2.6 Operators in the Bra–Ket Notation 183
9.2.7 Matrix Representations of Operators 185
9.3 The Spin Space of Single Noninteracting Spins 186
9.3.1 Expectation Values of the Spin-Components 188
9.4 Hamiltonian and Time Evolution 189
9.5 Free Precession 189
9.6 Representation of Spin Ensembles – The Density Matrix Formalism 191
9.6.1 Density Matrix at Thermal Equilibrium 193
9.6.2 Time Evolution of the Density Operator 193
9.7 Spin Systems 195
9.7.1 Scalar Coupling 196
Part Three Technical Aspects of NMR 199
10 The Components of an NMR Spectrometer 201
10.1 The Magnet 201
10.1.1 Field Homogeneity 202
10.1.2 Safety Notes 203
10.2 Shim System and Shimming 204
10.2.1 The Shims 204
10.2.2 Manual Shimming 205
10.2.3 Automatic Shimming 206
10.2.4 Using Shim Files 207
10.2.5 Sample Spinning 207
10.3 The Electronics 207
10.3.1 The RF Section 208
10.3.2 The Receiver Section 209
10.3.3 Other Electronics 209
10.4 The Probehead 209
10.4.1 Tuning and Matching 210
10.4.2 Inner and Outer Coils 211
10.4.3 Cryogenically Cooled Probes 211
10.5 The Lock System 212
10.5.1 The 2H Lock 212
10.5.2 Activating the Lock 213
10.5.3 Lock Parameters 214
10.6 Problems 214
Further Reading 214
11 Acquisition and Processing 215
11.1 The Time Domain Signal 217
11.2 Fourier Transform 219
11.2.1 Fourier Transform of Damped Oscillations 219
11.2.2 Intensity, Integral, and Line Width 220
11.2.3 Phases of Signals 221
11.2.4 Truncation 222
11.2.5 Handling Multiple Frequencies 222
11.2.6 Discrete Fourier Transform 223
11.2.7 Sampling Rate and Aliasing 224
11.2.8 How Fourier Transformation Works 225
11.3 Technical Details of Data Acquisition 229
11.3.1 Detection of the FID 229
11.3.2 Simultaneous and Sequential Sampling 230
11.3.3 Digitizer Resolution 233
11.3.4 Receiver Gain 234
11.3.5 Analog and Digital Filters 235
11.3.6 Spectral Resolution 236
11.4 Data Processing 237
11.4.1 Digital Resolution and Zero Filling 237
11.4.2 Linear Prediction 239
11.4.3 Pretreatment of the FID: Window Multiplication 240
11.4.4 Phase Correction 247
11.4.5 Magnitude Mode and Power Spectra 249
11.4.6 Baseline Correction 250
11.5 Problems 251
Further Reading 252
12 Experimental Techniques 253
12.1 RF Pulses 253
12.1.1 General Considerations 254
12.1.2 Hard Pulses 255
12.1.3 Soft Pulses 256
12.1.4 Band-Selective RF Pulses 257
12.1.5 Adiabatic RF Pulses 258
12.1.6 Composite Pulses 260
12.1.7 Technical Considerations 261
12.1.8 Sources and Consequences of Pulse Imperfections 263
12.1.9 RF Pulse Calibration 264
12.1.10 Transmitter Pulse Calibration 265
12.1.11 Decoupler Pulse Calibration (13C and 15N) 266
12.2 Pulsed Field Gradients 267
12.2.1 Field Gradients 267
12.2.2 Using Gradient Pulses 268
12.2.3 Technical Aspects 270
12.3 Phase Cycling 271
12.3.1 The Meaning of Phase Cycling 271
12.4 Decoupling 275
12.4.1 How Decoupling Works 275
12.4.2 Composite Pulse Decoupling 276
12.5 Isotropic Mixing 277
12.6 Solvent Suppression 277
12.6.1 Presaturation 278
12.6.2 Water Suppression through Gradient-Tailored Excitation 279
12.6.3 Excitation Sculpting 280
12.6.4 WET 280
12.6.5 One-Dimensional NOESY with Presaturation 280
12.6.6 Other Methods 281
12.7 Basic 1D Experiments 282
12.8 Measuring Relaxation Times 282
12.8.1 Measuring T1 Relaxation – The Inversion-Recovery Experiment 282
12.8.2 Measuring T2 Relaxation – The Spin Echo 283
12.9 The INEPT Experiment 286
12.10 The DEPT Experiment 288
12.11 Problems 290
13 The Art of Pulse Experiments 291
13.1 Introduction 291
13.2 Our Toolbox: Pulses, Delays, and Pulsed Field Gradients 292
13.3 The Excitation Block 293
13.3.1 A Simple 90 Pulse Experiment 293
13.3.2 The Effects of 180 Pulses 293
13.3.3 Handling of Solvent Signals 294
13.3.4 A Polarization Transfer Sequence 295
13.4 The Mixing Period 297
13.5 Simple Homonuclear 2D Sequences 298
13.6 Heteronuclear 2D Correlation Experiments 299
13.7 Experiments for Measuring Relaxation Times 301
13.8 Triple-Resonance NMR Experiments 303
13.9 Experimental Details 304
13.9.1 Selecting the Proper Coherence Pathway: Phase Cycles 304
13.9.2 Pulsed Field Gradients 306
13.9.3 N-Dimensional NMR and Sensitivity Enhancement Schemes 308
13.10 Problems 309
Further Reading 309
Part Four Important Phenomena and Methods in Modern NMR 311
14 Relaxation 313
14.1 Introduction 313
14.2 Relaxation: The Macroscopic Picture 313
14.3 The Microscopic Picture: Relaxation Mechanisms 314
14.3.1 Dipole–Dipole Relaxation 315
14.3.2 Chemical Shift Anisotropy 317
14.3.3 Scalar Relaxation 318
14.3.4 Quadrupolar Relaxation 318
14.3.5 Spin–Spin Rotation Relaxation 319
14.3.6 Paramagnetic Relaxation 319
14.4 Relaxation and Motion 319
14.4.1 A Mathematical Description of Motion:The Spectral Density Function 320
14.4.2 NMR Transitions That Can Be Used for Relaxation 322
14.4.3 The Mechanisms of T1 and T2 Relaxation 323
14.4.4 Transition Probabilities 324
14.4.5 Measuring Relaxation Rates 326
14.5 Measuring 15N Relaxation to Determine Protein Dynamics 326
14.5.1 The Lipari–Szabo Formalism 327
14.6 Measurement of Relaxation Dispersion 330
14.7 Problems 333
15 The Nuclear Overhauser Effect 335
15.1 Introduction 335
15.1.1 Steady-State and Transient NOEs 338
15.2 The Formal Description of the NOE: The Solomon Equations 338
15.2.1 Different Regimes and the Sign of the NOE: Extreme Narrowing and Spin Diffusion 340
15.2.2 The Steady-State NOE 341
15.2.3 The Transient NOE 344
15.2.4 The Kinetics of the NOE 344
15.2.5 The 2D NOESY Experiment 345
15.2.6 The Rotating-Frame NOE 347
15.2.7 The Heteronuclear NOE and the HOESY Experiment 349
15.3 Applications of the NOE in Stereochemical Analysis 350
15.4 Practical Tips for Measuring NOEs 352
15.5 Problems 353
Further Reading 354
16 Chemical and Conformational Exchange 355
16.1 Two-Site Exchange 355
16.1.1 Fast Exchange 358
16.1.2 Slow Exchange 360
16.1.3 Intermediate Exchange 360
16.1.4 Examples 362
16.2 Experimental Determination of the Rate Constants 364
16.3 Determination of the Activation Energyby Variable-Temperature NMR Experiments 366
16.4 Problems 368
Further Reading 369
17 Two-Dimensional NMR Spectroscopy 371
17.1 Introduction 371
17.2 The Appearance of 2D Spectra 372
17.3 Two-Dimensional NMR Spectroscopy: How Does It Work? 374
17.4 Types of 2D NMR Experiments 377
17.4.1 The COSY Experiment 378
17.4.2 The TOCSY Experiment 379
17.4.3 The NOESY Experiment 382
17.4.4 HSQC and HMQC Experiments 384
17.4.5 The HMBC Experiment 385
17.4.6 The HSQC-TOCSY Experiment 386
17.4.7 The INADEQUATE Experiment 387
17.4.8 J-Resolved NMR Experiments 388
17.5 Three-Dimensional NMR Spectroscopy 390
17.6 Practical Aspects of Measuring 2D Spectra 390
17.6.1 Frequency Discrimination in the Indirect Dimension:Quadrature Detection 390
17.6.2 Folding in 2D Spectra 396
17.6.3 Resolution in the Two Frequency Domains 397
17.6.4 Sensitivity of 2D NMR Experiments 398
17.6.5 Setting Up 2D Experiments 399
17.6.6 Processing 2D Spectra 400
17.7 Problems 401
18 Solid-State NMR Experiments 403
18.1 Introduction 403
18.2 The Chemical Shift in the Solid State 404
18.3 Dipolar Couplings in the Solid State 406
18.4 Removing CSA and Dipolar Couplings: Magic-Angle Spinning 407
18.5 Reintroducing Dipolar Couplings under MAS Conditions 408
18.5.1 An Alternative to Rotor-Synchronized RF Pulses: Rotational Resonance 410
18.6 Polarization Transfer in the Solid State: Cross-Polarization 411
18.7 Technical Aspects of Solid-State NMR Experiments 413
18.8 Problems 414
Further Reading 414
19 Detection of Intermolecular Interactions 415
19.1 Introduction 415
19.2 Chemical Shift Perturbation 417
19.3 Methods Based on Changes in Transverse Relaxation(Ligand-Observe Methods) 418
19.4 Methods Based on Changes in Cross-Relaxation (NOEs) (Ligand-Observe or Target-Observe Methods) 420
19.5 Methods Based on Changes in Diffusion Rates(Ligand-Observe Methods) 423
19.6 Comparison of Methods 424
19.7 Problems 425
Further Reading 426
Part Five Structure Determination of Natural Products by NMR 427
20 Carbohydrates 439
20.1 The Chemical Nature of Carbohydrates 439
20.1.1 Conformations of Monosaccharides 442
20.2 NMR Spectroscopy of Carbohydrates 443
20.2.1 Chemical Shift Ranges 443
20.2.2 Systematic Identification by NMR Spectroscopy 444
20.2.3 Practical Tips: The Choice of Solvent 449
20.3 Quick Identification 450
20.4 A Worked Example: Sucrose 450
Further Reading 457
21 Steroids 459
21.1 Introduction 459
21.1.1 The Chemical Nature 460
21.1.2 Proton NMR Spectra of Steroids 461
21.1.3 Carbon Chemical Shifts 463
21.1.4 Assignment Strategies 464
21.1.5 Identification of the Stereochemistry 467
21.2 A Worked Example: Prednisone 469
Further Reading 476
22 Peptides and Proteins 477
22.1 Introduction 477
22.2 The Structure of Peptides and Proteins 478
22.3 NMR of Peptides and Proteins 481
22.3.1 1H NMR 481
22.3.2 13C NMR 484
22.3.3 15N NMR 487
22.4 Assignment of Peptide and Protein Resonances 489
22.4.1 Peptides 490
22.4.2 Proteins 493
22.5 A Worked Example: The Pentapeptide TP5 496
Further Reading 500
23 Nucleic Acids 501
23.1 Introduction 501
23.2 The Structure of DNA and RNA 502
23.3 NMR of DNA and RNA 506
23.3.1 1H NMR 506
23.3.2 13C NMR 509
23.3.3 15N NMR 510
23.3.4 31P NMR 510
23.4 Assignment of DNA and RNA Resonances 512
23.4.1 Unlabeled DNA/RNA 512
23.4.2 Labeled DNA/RNA 516
Further Reading 518
Appendix 519
A.1 The Magnetic H and B Fields 519
A.2 Magnetic Dipole Moment and Magnetization 520
A.3 Scalars, Vectors, and Tensors 521
A.3.1 Properties of Matrices 524
Solutions 527
Index 545
1
Introduction to NMR Spectroscopy
Tremendous progress has been made in NMR spectroscopy with the introduction of multidimensional NMR spectroscopy and pulse Fourier transform NMR spectroscopy. For a deeper understanding of the experiment, a little knowledge of quantum physics is required. We will summarize the physical foundations of NMR spectroscopy in more detail in the following chapter. In this chapter, we will introduce the novice reader to the field of NMR spectroscopy in a simple way like we ourselves were introduced to it a long time ago. We will show some simple 1D spectra, and briefly describe what kind of information we can extract from these. For the moment we will assume that the spectra have been recorded by “someone,” and we will skip the technical aspects. Later in the book we will discuss all aspects of NMR spectroscopy – experimental, technical, and theoretical – to make you an NMR expert, who can run your own spectra and interpret them skillfully. You should then also have obtained the necessary knowledge for troubleshooting problems during data acquisition. Throughout the book we will introduce you to a subject first in a simple way, and then extend the discussion to more specialized topics and provide a more rigorous explanation.
1.1 Our First 1D Spectrum
Let us jump right into cold water and have a first glimpse at the spectrum of a simple organic compound. As an example we will choose an aromatic compound that is a natural product but produced synthetically on a large scale, called vanillin. So, let us have a first look at the proton spectrum (Figure 1.1).
Figure 1.1 Proton NMR spectrum of a simple organic compound. The two arrows point to the standard for referencing (the tetramethylsilane signals) and the solvent line (the dimethyl sulfoxide signal). Integral traces are depicted above the signals. The expansion shows the aromatic protons.
We notice a number of signals at various places. The signals seem to be of different intensity. If we look a bit more closely, we recognize that lines are split into multiplets (see the expansion). Below the spectrum we find a scale which roughly runs from 0 to 10 ppm. The signals indicated by an arrow belong to the solvent (the signal at 2.5 ppm is from residual dimethyl sulfoxide and the signal at 0 ppm is from the tetramethylsilane standard used for referencing). Otherwise we can count six signals, corresponding to six different types of protons in vanillin. The region from 6.9 to 7.5 ppm is expanded in the top panel. To start, let us learn a bit of nomenclature first
1.2 Some Nomenclature: Chemical Shifts, Line Widths, and Scalar Couplings
The phenomenon that the resonance frequency of a nucleus depends on the chemical environment is called chemical shift.1) The chemical shift is largely determined by the electron density around the nucleus. For practical reasons the chemical shift is given in parts per million relative to a standard. Chemical shifts, in general, are an invaluable source of information for the interpretation of spectra. In principle, they can be computed fairly precisely nowadays using quantum mechanical methods such as density functional theory. What makes chemical shifts really useful is that they are influenced by the presence of functional groups, double bonds, aromatic ring systems, and so on. This has led to elaborate tables of chemical shifts empirically derived from databases. You will find many of these tables in our chapters on proton and heteronuclear NMR, or in textbooks dedicated to that purpose. As a chemist, however, you will need to “memorize” some basic values. If you are working on a certain class of compounds, you will become an expert on chemical shifts for these molecules.
Let us now look more closely at a single line (Figure 1.2).
Figure 1.2 (a) A single resonance line. The frequency scale runs from the right to the left. A line with typical Lorentzian shape is depicted in (b).
The line has a certain shape, a Lorentzian lineform. The signal is symmetric, and the highest intensity denotes the chemical shift position δ. The line width of the signal usually refers to the width at half height. Increasing values of chemical shift or frequency are plotted to the left for traditional reasons (note this is different from how it is usually done in physics or mathematics). Although the signals occur at certain frequencies, the frequency scale itself is not drawn, because it depends on the strength of the magnet. Instead, the values are presented in parts per million, which is the difference in frequency from a standard normalized by the frequency of the standard (do not worry, we will see how this scale is derived in more detail later).
Often signals are split into a number of lines (Figure 1.3), sometimes as many as nine or even more. These splittings are called scalar couplings, and originate from an interaction of the corresponding proton with neighboring protons, either on the same carbon or on the adjacent carbon(s) or heteroatom.
Figure 1.3 Scalar J couplings. Typical multiplet patterns for doublets, triplets and quartets are shown.
The center of the multiplet corresponds to the chemical shift δ of that signal. The separation of adjacent lines is called the scalar coupling constant, often abbreviated as J. Depending on whether the neighboring carbons are separated by rotatable bonds or whether the bond is sterically fixed, the number of lines due to scalar coupling is N + 1 (free rotation about the C–C bond) or 2N (defined dihedral angle), where N denotes the number of neighboring protons. J is independent of the magnetic field strength and is specified in hertz. The individual lines often have different intensities (see Figure 1.3). Shown on the right of Figure 1.3 is a singlet, a doublet, a triplet, and a quartet. In the case of the quartet, the line intensities are 1 : 3 : 3 : 1. Since the number of lines follows simple rules, it helps us to establish the environment of the proton.
The intensity of the signals can be determined by integrating the spectra, and the integrals will tell us whether a certain signal is due to one, two, three, or more protons (Figure 1.4).
Figure 1.4 The effect of variable line widths. Two lines of very different intensity but the same integral are shown.
Integrals can be drawn as integral trails (usually directly on top of the signal) or their value can be plotted below the signal. Figure 1.4 displays two signals of identical integral but very different line width, with the signal at the lower frequency (the one on the right) being less intense. The line width has diagnostic value that is often underappreciated. Some lines become broader than others because the lifetime of the proton in a certain environment is short, a phenomenon due to either chemical or conformational exchange.
Spectra often also contain lines that do not belong to the molecule under study; some of them are referred to as artifacts. Such signals can belong to the solvent. In Fourier transform NMR spectroscopy deuterated solvents are mandatory, but the degree of deuteration is never 100% and residual signal from the nondeuterated form is present. Another signal that is almost always present in proton spectra is the signal due to water, either from residual water in the solvent or because the compound has not been dried completely. Thirdly, a standard is often added for calibrating spectra. In most organic solvents tetramethylsilane is used because the signal usually occurs at one end of the spectrum and does not overlap with the signals of interest. Two-dimensional spectra contain other artifacts that are due to incomplete removal of unwanted coherence pathways, and we will deal with them later.
1.3 Interpretation of Spectra: A Simple Example
To get used to interpreting spectra, and to illustrate the strength of NMR spectroscopy, let us try to elucidate the structure of a small organic molecule. Its 1H spectrum is shown in Figure 1.5.
Figure 1.5 Proton NMR spectrum of ibuprofen.
The spectrum displays a number of signals, and the particular location of the signals, the chemical shift, already tells us a lot about the chemical nature of this molecule. For example, the signals at 7 ppm appear in a range that is typical for aromatic protons. Or, the signal around 3.6 ppm is most likely from a proton in the vicinity of some heteroatom. The signals around 1 ppm are most likely from methyl protons, which is also supported by the integral values of 3 and 6, respectively. Even more helpful is the fine structure of the signals. To see that, let us zoom in a bit on the spectrum (Figure 1.6).
Figure 1.6 Expansions of the proton NMR spectrum revealing the multiplet fine structure of the signals.
Most of the signals display the usual (N + 1) multiplet pattern expected for protons in freely rotatable chains. The signal group labeled with 6 in Figure 1.6 consists of two doublets, which however, for reasons which will be explained in Section 3.4.2, are somewhat skewed. So let us begin building up the molecule.
We start with the signal group 6 in the range from 7–7.2 ppm. As mentioned before, this is the range typically observed for aromatic protons. The integral of these signals corresponds to 4. Although we do not know much about the chemical nature...
| Erscheint lt. Verlag | 26.11.2013 |
|---|---|
| Sprache | englisch |
| Themenwelt | Naturwissenschaften ► Chemie |
| Naturwissenschaften ► Physik / Astronomie | |
| Technik | |
| Schlagworte | applicationoriented • Applications • behind nmr • Biochemie • Biochemie u. Chemische Biologie • biochemistry • Biochemistry (Chemical Biology) • biomolecular • Biowissenschaften • Chemical • Chemie • Chemistry • Complex • Elucidation • general description • important • introducing • Life Sciences • Modern • NMR • NMR Spectroscopy / MRI / Imaging • NMR-Spektroskopie • NMR-Spektroskopie / MRT / Bildgebende Verfahren • parameters • Part • Pharmaceutical & Medicinal Chemistry • Pharmazeutische u. Medizinische Chemie • Physics • Practice • Profound • shorter • Spektroskopie • Spin • stage • Structure • theory |
| ISBN-10 | 3-527-67785-2 / 3527677852 |
| ISBN-13 | 978-3-527-67785-6 / 9783527677856 |
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Details zum Adobe-DRM
Dateiformat: EPUB (Electronic Publication)
EPUB ist ein offener Standard für eBooks und eignet sich besonders zur Darstellung von Belletristik und Sachbüchern. Der Fließtext wird dynamisch an die Display- und Schriftgröße angepasst. Auch für mobile Lesegeräte ist EPUB daher gut geeignet.
Systemvoraussetzungen:
PC/Mac: Mit einem PC oder Mac können Sie dieses eBook lesen. Sie benötigen eine
eReader: Dieses eBook kann mit (fast) allen eBook-Readern gelesen werden. Mit dem amazon-Kindle ist es aber nicht kompatibel.
Smartphone/Tablet: Egal ob Apple oder Android, dieses eBook können Sie lesen. Sie benötigen eine
Geräteliste und zusätzliche Hinweise
Zusätzliches Feature: Online Lesen
Dieses eBook können Sie zusätzlich zum Download auch online im Webbrowser lesen.
Buying eBooks from abroad
For tax law reasons we can sell eBooks just within Germany and Switzerland. Regrettably we cannot fulfill eBook-orders from other countries.
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