1 H- Nuclear Magnetic Resonance

It is spectroscopy technique to determine number of hydrogen atoms present in the molecules and atoms.It is useful method for separation of molecules and compounds from mixtures components highly recommended in pharmaceutical and chemical engineering fields.

1. Nuclear Magnetic Resonance (NMR) Spectroscopy 9/3/2021 1 PREPARED BY:-AJAY KUMAR

2. Felix Bloch 1905-1983 Edward M. Purcell 1912-1997 Kurt Wuthrich 1938- Richard R. Ernst 1933-(Nobel Prize in 1991) CW NMR 40MHz 1960 9/3/2021 9/3/2021

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4. A. Chemical-related research: Analytical tool. Structural characterization of chemical compounds. B. Material-related research Polymer characterization. C60 (Fullerene). High temperature superconductor research. Heterogeneous catalysis (Ziolite). Surface physics. C. Study of dynamic processes Reaction kinetics. Study of equilibrium (chemical or structural). D. Structural (three-dimensional) studies Proteins. DNA/RNA. Protein complexes with DNA/RNA. Polysaccharides Why bother learning NMR? 9/3/2021 4

5. E. Drug design Structure Activity Relationships (SAR) by NMR F. Biomedical applications: Metabolic studies of biological systems. Magnetic Resonance Imaging (MRI), diagnostic imaging, flow imaging, chemical shift imaging, functional imaging. Macromolecular structure determination in solution. Finally , it’s the biggest, meanest, most expensive piece of equipment you’ll see in your career, and this is a great time to get your hands on it... Why bother learning NMR? 9/3/2021 5

6. 9/3/2021 6 NMR Historic Review 1924 Pauli proposed the presence of nuclear magnetic moment to explain the hyperfine structure in atomic spectral lines. 1930 Nuclear magnetic moment was detected using refined Stern- Gerlach experiment by Estermann . 1939 Rabi et al. First detected unclear magnetic resonance phenomenon by applying r.f . energy to a beam of hydrogen molecules in the Stern- Gerach set up and observed measurable deflection of the beam. In 1944 Rabi awarded Nobel prize in physics 1946 Purcell et al. at Harvard reported nuclear resonance absorption in paraffin wax. Bloch et al. at Stanford found nuclear resonance in liquid water. 1949 Chemical shift phenomenon was observed. 1952 Nobel prize in Physics was awarded to Purcell and Bloch, first practical NMR experiments, which were carried out independently by both of them in 1945 at different places. 1960 Ernst and Anderson first introduce the Fourier Transform technique into NMR

7. 9/3/2021 7 Late in 1960 Solid State NMR was revived due to the effort of Waugh. and associates at MIT. Biological application become possible due to the introduction superconducting magnets. NMR imaging was demonstrated. 1970 2D NMR was introduced 1980s Macromolecular structure determination in solution by NMR was achieved. 1991 Nobel prize in Chemistry was awarded to Richard Ernst for he developed Fourier transformation method 1990s Continuing development of heteronuclear multi-dimensional NMR permit the determination of protein structure up to 50 KDa . MRI become a major radiological tool in medical diagnostic. 2002 Nobel prize in Chemistry was awarded to Kurt Wuthrich for the elucidation of three-dimensional structures of macromolecules. 2003 Nobel Prizes were awarded to Lauterbach and Mansfield for their research in magnetic resonance imaging. (MRI) NMR Historic Review

8. 9/3/2021 Nuclear Spin explanation The spinning charged nucleus generates a magnetic field. => 8

9. 9/3/2021 Protons (and other nucleons) have Spin Spin up (+1/2) Spin down (-1/2) 9

10. 9/3/2021 Each Spinning Proton is Like a “Mini-Magnet” Spin up Spin down N S N S 10

11. The nucleus in free state 9/3/2021 11

12. Nuclear spins 9/3/2021 12

13. 9/3/2021 Principles of NMR The nucleus spin on its own axis and magnetic moment is created, resulting in the precessional orbit with a frequency called as processional frequency. The nucleus of hydrogen atom behaves as spin bar magnet because it possess both electric and magnetic field. NMR involves the interaction between an oscillating magnetic field of EMR and the magnetic energy of the hydrogen nucleus or some other nuclei, when they are placed in external magnetic field. 13

14. In any magnetic field, magnetic nuclei like proton precess at a frequency, v which is proportional to the strength of the applied field. The exact frequency is expressed by v=µ  N B o / h I Where Bo =strength of the external field experienced by the proton I=Spin quantum number h=Planks constant (6.626x10 -34 Js) µ=Magnetic moment of the particular nucleus  N = Nuclear magnet on constant L= angular momentum associated with the nuclei We can think of nuclei as small magnetized tops that spin on their axis: The magnetic nuclei has two forces acting on the spins. One that tries to turn them towards B o , and the other that wants to maintain their angular momentum. The net result is that the nuclei spins like a top B o w o m L m L 9/3/2021 14 9/3/2021 Precessional Frequency or Larmor frequency

15. Precession (continued) Spins won’t align with B o , no matter what their intiial orientation was. Spins pointing ‘up’ and ‘down’ don’t exist! Spins will precess at the angle they were when we turned on the magnetic field B o : B o There are several magnetic fields acting on the spins. One is Bo , which is constant in time and generates the precession at w o . The others are fluctuating due to the molecular anisotropy and its environment, and make the spins ‘try’ all the possible orientations with respect to B o in a certain amount of time. Orientations in favor of Bo will have lower magnetic energy, and will be slightly favored. 9/3/2021 15 9/3/2021

16. 9/3/2021 Principles of NMR All nuclei in a molecule are surrounded by electron clouds H effective= H applied - H local The local magnetic field can either reinforce the applied magnetic field, deshield the hydrogen nucleus, and hence a higher frequency will be required to bring it into resonance. The local magnetic field can oppose the applied magnetic field, shield the hydrogen nucleus from the strength of the magnetic field and hence a lower frequency will be required to bring it into resonance. Thus slightly different amounts of energy are needed to excite each individual hydrogen nucleus to its own higher energy level. In summary, different electron densities create different magnetic environments around each hydrogen atom and therefore a series of signals are seen across a spectrum. 16

17. A nucleus with an odd atomic number or an odd mass number has a nuclear spin. The angular momentum of the charge is described as spin number. They have 0, ½,1, 3/2…etc (I =0 denotes no spin) Each proton and neutron has its own spin. If sum of the proton and neutron is even, spin number (I)= 0,1,2,3, etc If sum of the proton and neutron is odd I is half integral=1/2, 3/2, 5/2…etc Principles of NMR 9/3/2021 17

18. All nuclei carry a charge and in some nuclei this charge spins around an axis generating a magnetic dipole along the axis of the nucleus. 1 H has a spin I = ½. There are two allowed spin states –½ and +½. In the absence of a magnetic field the two spin states are degenerate and are equally populated. In a magentic field the low energy spin state is aligned with the magnetic field and the high energy opposed to it. Principles of NMR 9/3/2021 18

19. The case of 1/2 spin nuclei. Presence of external field orients the spins. Spins that are opposed to the field have higher energy than spins that are aligned with the field. Principles of NMR 9/3/2021 19

20. 9/3/2021 NMR equation Energy difference is proportional to the magnetic field strength.  E = h =  h B 2 Gyromagnetic ratio, , is a constant for each nucleus (26,753 s -1 gauss -1 for H). In a 14,092 gauss field, a 60 MHz photon is required to flip a proton. Low energy, radio frequency. => 20

21. The sample absorb different EMR at different frequency. The spinning axis of the top moves slowly around the vertical. It has been found that the proton precesses about the axis of the external magnetic field It has been found that w =  H o --------(1) Where w= angular precessional velocity H o =applied field in gauss  = Gyromagnetic ratio = 2  hI 9/3/2021 21

22. Here  =magnetic movement of the spinning bar magnet I = is the spin quantum number of the spinning magnet h= Planck’s constant According to the fundamental NMR equation which correlates EMR frequencies with the magnetic field we say that  H o =2  ------------(2) Here v is the frequency of EMR From equation 1) and 2) Angular precessional velocity w = 2  v 9/3/2021 22

23. Precessional frequency –No. of revolution/sec. made by the magnetic movement vector of the nucleus around the external field H o Alternatively it is defined as equal to the frequency of EMR in megacycles per second necessary to induce transition from one spin state to another. All nuclei carry a charge , so they will possess spin angular- momentum. The nuclei which have a finite value of spin quantum number (I >0) will precess along the axis of rotation. 9/3/2021 23

24. Nuclear spin is the total nuclear angular momentum quantum number. This is characterized by a quantum number I , which may be integral, half-integral or 0. Only nuclei with spin number I  0 can absorb/emit electromagnetic radiation. The magnetic quantum number m I has values of –I, -I+1, …..+I ( e.g. for I=3/2, m I =-3/2, -1/2, 1/2, 3/2 ). 1. A nucleus with an even mass A and even charge Z  nuclear spin I is zero. Example: 12 C, 16 O, 32 S  No NMR signal 2. A nucleus with an even mass A and odd charge Z  integer value I . Example: 2 H, 10 B, 14 N  NMR detectable 3. A nucleus with odd mass A  I=n/2, where n is an odd integer. Example: 1 H, 13 C, 15 N, 31 P  NMR detectable Properties of the Nucleus 9/3/2021 24

25. To observe resonance, we have to irradiate the molecule with EMR of the appropriate frequency. Different nucleus “type” will give different NMR signal. D epending on the chemical environment, there are variations on the magnetic field that the nuclei feels , even for the same type of nuclei. The main reason for this is, each nuclei could be surrounded by different electron environment, which make the nuclei “feel” different net magnetic field 9/3/2021 25 NMR signals

26. If the oriented nuclei are now irradiated with EMR of proper energy, frequency absorption occurs and the lower energy spin flips to higher energy state. When this spin flip occurs, the nuclei are said to be in Resonance with applied radiation, hence named NMR. The exact amount of RF energy necessary for resonance depends on the strength of external magnetic field and the nuclei being irradiated. Strong magnetic field higher energy Weaker magnetic field less energy 9/3/2021 26

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28. 9/3/2021 28 C C

29. 9/3/2021 Magnetic Shielding If all protons absorbed the same amount of energy in a given magnetic field, not much information could be obtained. But protons are surrounded by electrons that shield them from the external field. Circulating electrons create an induced magnetic field that opposes the external magnetic field. => 29

30. 9/3/2021 Shielded Protons Magnetic field strength must be increased for a shielded proton to flip at the same frequency. 30

31. 9/3/2021 Protons in a Molecule Depending on their chemical environment, protons in a molecule are shielded by different amounts. => 31

32. At 60MHZ 1MHZ= 1 million cycles per second to bring 1 H nucleus to resonate 15 MHZ required to bring 13 C nucleus to resonate These energy comparatively less than for which is needed in IR For IR 1.1-11K.calories / mol. NMR 5.7x 10 -6 K.calories / mol. Energy used in NMR 9/3/2021 32

33. 9/3/2021 Energy used in NMR E m = energy of quantum state m (J)  = gyromagnetic ratio (T –1 s –1 ) h = Planck’s constant (6.628  10 –34 J s) B = applied magnetic field (T  Tesla ) 33

34. 9/3/2021 What factors affect sensitivity? To enhance sensitivity, Increase magnetic field strength, B Choose nucleus with large gyromagnetic ratio,  Carry out experiment at low temperature, T 34

35. The chemical shift The resonant frequency of a certain atom is called chemical shift. Advantages: More compact annotations Independent on the spectrometer field In practice, the 1 H chemical shifts are in the range 0-10 ppm or  The chemical shift depends on: The atom type (NH, aliphatic CH, aromatic CH, ...) The amino acid type (Ala, Phe , ...) The chemical (spatial) environment 9/3/2021 35

36. 9/3/2021 NMR Signals The number of signals shows how many different kinds of protons are present. The location of the signals shows how shielded or deshielded the proton is. The intensity of the signal shows the number of protons of that type. Signal splitting shows the number of protons on adjacent atoms. To define the position of absorption the NMR chart is calibrated by using TMS highly shielded molecule. The exact place on the chart at which a nucleus absorb is called “chemical shift” 36

37. Chemical shift of TMS is arbitrarily set the zero point 1 = 1ppm of the spectrometer operating frequency 1 H NMR, 60MHz operating instrument 60,000,000 1 = 1ppm (or) 60Hz 100MHZ operating instrument 1 = 100Hz  (ppm)= observed chemical shift (no.of Hz away from TMS) Spectrometer frequency in MHz If the induced field, opposes the applied field the electrons are diamagnetic & the effect is diamagnetic shielding. 9/3/2021 37

38. 9/3/2021 Types of NMR Continuous wave ( cw ) NMR It detects the resonance of nuclei FT NMR Directly recording the intensity of absorption as a function of frequency The magnet used in NMR produces strong magnetic field sample probe 1) Sample probe contained between the poles of the magnet 38

39. 9/3/2021 The probe has another coil wrapped at right angles to the transmitter coil Thus the optimum angle for detecting resonance Various temp probe helps to keep the sample at different temp (-100 to 200°C) So NMR can be used for kinetic study, thermodynamics 39

40. 9/3/2021 40 The chemical shift (δ) is defined as the difference between the resonance position of a sample nucleus and that of a standard TMS. Chemical shift ( δ) = [ Δν ( Hz)/ Applied resonance frequency × 10 6 Hz)] × 10 6 ppm where, Δν = Difference in frequency (Hz) between the observed signal and that of the standard. Convention for δ : TMS assigned (δ = 0), values for other protons are measured positively downfield. In other words, increasing δ corresponds to increasing de-shielding of the nucleus.

41. 9/3/2021 41 Spin-spin Interactions High resolution NMR spectra very often exhibit signals as multiplets , invariably showing a more or less symmetrical appearance. Multiplicity is brought about due to the splitting of the signal of one set of equivalent nuclei by the magnetic fields of adjacent sets of nuclei i.e., spin-spin interactions.

42. 9/3/2021 42 Block Diagram of NMR Spectrometer

43. 9/3/2021 Radiofrequency Oscillator It generated by electronic multiplication and natural frequency of quartz crystal contained in a thermostated block/different crystal/trasmitters are used RFO installed perpendicular to the magnetic field & transmits RW of fixed frequency 60,100, 200, 300 & 500 MHZ 1 MHZ is =1 million cycles per second 43

44. 9/3/2021 RF receiver/detector: The sample in the NMR probe gives data which can be detected as a signal Detectors used in NMR should be sensitive as the signal levels are small(<1milli volt) So that multiplication of signal is essential Rf receiver also to be perpendicular to the magnet like oscillator 44

45. 9/3/2021 Recorder The signal is sent to the recorder or oscilloscope It helps fast scanning of spectrum or electronic filtering of signal The recorder plots resonance signal on y-axis and strength of magnetic field on x-axis The strength of resonance signal  number of nuclei resonating at that field strength Most spectrometer equipped with automatic integrator to measure the area under the observed signal 45

46. 9/3/2021 Solvents CDCl 3 Advantages for non-polar to polar compounds CDCl 3 peak appear in 7.27 δ for sparingly soluble compounds add drop wise (DMSO-d6) It will shift residual CHCl 3 peak to 8.38 δ CCl 4 for non polar compounds any water molecule causes turbidity DMSO-d6 More viscous/restricted rotation, causes line broadening non-volatile (difficult to remove from the sample) 46

47. 9/3/2021 Additional Factors Affecting Chemical Shift It is very difficult to predict the chemical shift of protons attached to heteroatoms (O-H, N-H, S-H). It is due to hydrogen bonding which has the effect deshielding the proton and it causes broadening of signal. Replaces the exchangeable protons with deuteriums which cannot be detected in the proton NMR. Hence its chemical shift can be identified without any problem (It is a useful technique for -OH, NH 2 and COOH 47

48. 9/3/2021 Solvent effect Changing the solvent has a dramatic, yet unpredictable effect on the chemical shift of signals. This is useful if important peaks overlap in a CDCl 3 spectrum then another solvent, D 6 benzene, D 3 acetonitrile etc can be used 48

49. Chemical Shift Data Different kinds of protons typically come at different chemical shifts. Shown below is a chart of where some common kinds of protons appear in the delda scale. Note that most protons appear between 0 and 10 ppm. The reference, tetramethylsilane (TMS) appears at 0 ppm, and aldehydes appear near 10 ppm. 9/3/2021 49

50. 9/3/2021 Location of Signals More electronegative atoms deshield more and give larger ᵟ values. Effect decreases with distance. Additional electronegative atoms cause increase in chemical shift. 50

51. 9/3/2021 Typical Values => 51

52. 9/3/2021 Aromatic Protons, 7-8 52

53. 9/3/2021 Vinyl Protons, 5-6 => 53

54. 9/3/2021 Acetylenic Protons, 2.5 => 54

55. 9/3/2021 Aldehyde Proton,  9-10 => Electronegative oxygen atom 55

56. 9/3/2021 O-H and N-H Signals Chemical shift depends on concentration. Hydrogen bonding in concentrated solutions deshield the protons, so signal is around  3.5 for N-H and  4.5 for O-H. Proton exchanges between the molecules broaden the peak. 56

57. 9/3/2021 Carboxylic Acid Proton, 10+ => 57

58. 9/3/2021 Number of Signals Equivalent hydrogens have the same chemical shift. => 58

59. 9/3/2021 Intensity of Signals The area under each peak is proportional to the number of protons. Shown by integral trace. => 59

60. H O-C H 2 -C H 3 low field high field Notice that the intensity of peak is proportional to the number of H 9/3/2021 60

61. 1H 13C Example of 1D : 1H spectra, 13C spectra of Codeine C 18 H 21 NO 3 , MW= 299.4 9/3/2021 61

62. 9/3/2021 How Many Hydrogens ? When the molecular formula is known, each integral rise can be assigned to a particular number of hydrogens. => 62

63. The Hard Part - Interpreting Spectra Following is the NMR spectrum of ethyl acetate. Since each NMR spectrum is a puzzle What kinds of data do we get from NMR spectra? For 1 H NMR, there are three kinds each of which we will consider each of these separately: 1) Chemical shift data - tells us what kinds of protons we have. 2) Integrals - tells us the ratio of each kind of proton in our sample. 3) 1H - 1H coupling - tells us about protons that are near other protons. 9/3/2021 63

64. Integrals Integrals tell us the ratio of each kind of proton. They are lines, the heights of which are proportional to the intensity of the signal. Example (ethyl acetate) three kinds of protons - CH 3 next to the carbonyl, CH 2 next to the O and the CH 3 next to the CH 2 . The ratio of the (height) signals arising from each of these kinds of protons should be 3 to 2 to 3, respectively. This will help us to identify CH 2 signal (it’s the smallest one), but to distinguish the other two, we have to be able to predict their chemical shifts. T The CH 3 next to the C=O should appear at ~ 2 PPM, while the other CH 3 should be at ~ 1 PPM). 9/3/2021 64

65. 9/3/2021 Spin-Spin Splitting Nonequivalent protons on adjacent carbons have magnetic fields that may align with or oppose the external field. This magnetic coupling causes the proton to absorb slightly downfield when the external field is reinforced and slightly up field when the external field is opposed. All possibilities exist, so signal is split. 65

66. 9/3/2021 1,1,2-Tribromoethane Nonequivalent protons on adjacent carbons. => 66

67. 9/3/2021 Doublet: 1 Adjacent Proton => 67

68. 9/3/2021 Triplet: 2 Adjacent Protons => 68

69. 9/3/2021 The N + 1 Rule If a signal is split by N equivalent protons, it is split into N + 1 peaks. => 69

70. Splitting Patterns with Multiple Neighboring Protons If a proton has n neighboring protons that are equivalent , that proton will be split into n+1 lines. So, if we have four equivalent neighbors, we will have five lines, six equivalent neighbors… well, you can do the math. The lines will not be of equal intensity, rather their intensity will be given by Pascal’s triangle as shown below. We keep emphasizing that this pattern only holds for when the neighboring protons are equivalent. Why is that? The answer is two slides away. 9/3/2021 70

71. 9/3/2021 Splitting for Ethyl Groups => 71

72. 9/3/2021 Splitting for Isopropyl Groups => 72

73. 9/3/2021 Values for Coupling Constants => 73

74. 9/3/2021 Complex Splitting Signals may be split by adjacent protons, different from each other, with different coupling constants. Example: H a of styrene which is split by an adjacent H trans to it (J = 17 Hz) and an adjacent H cis to it (J = 11 Hz). => 74

75. 9/3/2021 Coupling Constant (J) It represents regular multiplets . Actually, J is the separation (in Hertz ; Hz = sec–1) between the peaks of regular multiplets . The coupling constants help in the identification of the coupled nuclei because Jac = Jca : and are therefore, useful in characterizing the relative orientations of interacting protons.

76. 9/3/2021 Splitting Tree => 76

77. 9/3/2021 Spectrum for Styrene => 77

78. 9/3/2021 Stereochemical Nonequivalence Usually, two protons on the same C are equivalent and do not split each other. If the replacement of each of the protons of a -CH 2 group with an imaginary “Z” gives stereoisomers, then the protons are non-equivalent and will split each other. => 78

79. 9/3/2021 Some Nonequivalent Protons => 79

80. 9/3/2021 Hydroxyl Proton Ultrapure samples of ethanol show splitting. Ethanol with a small amount of acidic or basic impurities will not show splitting. => 80

81. 9/3/2021 Cl CH 2 OH CH 2 3.4- 4.0 and for OH 4.0-5.0 2+1 protons (no splitting) Acetaldehyde CH 3 2.1 -2.3 and for CHO 9.5-10.1 3+1 protons(no splitting) N-Pentane CH 3 0.8-1.0 (2+1 protons -triplet) and CH 2 1.2-1.4 (multiplet) Benzene CH 6.5- 8.5 (no splitting) Toluene CH 3 2.2-2.5 and for CH 6.5-8.5 (no splitting)

82. 9/3/2021 N-H Proton Moderate rate of exchange. Peak may be broad. => 82

83. 9/3/2021 Identifying the O-H or N-H Peak Chemical shift will depend on concentration and solvent. To verify that a particular peak is due to O-H or N-H, shake the sample with D 2 O Deuterium will exchange with the O-H or N-H protons. On a second NMR spectrum the peak will be absent, or much less intense. 83

84. 9/3/2021 Spin-Spin Coupling Many 1 H NMR spectra exhibit peak splitting (doublets, triplets, quartets) This splitting arises from adjacent hydrogens (protons) which cause the absorption frequencies of the observed 1 H to jump to different levels These energy jumps are quantized and the number of levels or splittings = n+1 where “n” is the number of nearby 1 H’s 84

85. 9/3/2021 Spin-Spin Coupling C - Y C - CH C - CH 2 C - CH 3 H | H | H | H | singlet doublet triplet quartet X Z X Z X Z X Z J 85

86. 9/3/2021 Spin Coupling Intensities 1 1 1 1 2 1 1 3 3 1 1 4 6 4 1 1 5 10 10 5 1 1 1 1 1 2 Pascal’s Triangle 1 1 3 3 86

87. 9/3/2021 NMR Peak Intensities C - C H C - C H 2 C - C H 3 Y | Y | Y | X Z X Z X Z AUC = 1 AUC = 2 AUC = 3

88. 9/3/2021 Low Resolution NMR Spectrum of Ethanol, CH 3 CH 2 OH Chemical shift, d = ( n r - n s ) × 10 6 (ppm)

89. 9/3/2021 High Resolution NMR Spectrum of Ethanol, CH 3 CH 2 OH Why?

90. 9/3/2021 Modes of NMR Relaxation Spin-lattice (longitudinal) relaxation characterized by relaxation time T 1 Spin-spin (transverse) relaxation characterized by relaxation time T 2 T 1 and T 2 are typically 0.1-1.0 s

91. 9/3/2021 Spin Lattice Relaxation Caused by random fluctuations of nuclei in sample, whose moving magnetic field can induce transitions in magnetic moment of observed nucleus T 1 is large in solids, much smaller in liquids T 1 is very short in the presence of paramagnetic species, or m >½ nuclei

92. 9/3/2021 Spin-Spin Relaxation Caused by interaction between nuclei having the same Larmor frequency, but different energy states Results in dephasing of precessing nuclei, causing line-broadening of observed nucleus

93. Spin-Lattice Coupling (Nuclear Overhauser Effect) Two nuclear spins within about 5 Å will interact with each other through space. This interaction is called cross-relaxation, and it gives rise to the nuclear Overhauser effect (NOE). Two spins have 4 energy levels, and the transitions along the edges correspond to transitions of one or the other spin alone. W 2 and W are the cross-relaxation pathways, which depend on the tumbling of the molecule. Nuclear spins can also cross-relax through dipole-dipole interactions and other mechanisms. This cross relaxation causes changes in one spin through perturbations of the other spin. Intensity of the NOE is proportional to r -6 ( r is distance between 2 spins). 9/3/2021 93

94. Spin-Lattice Coupling (Nuclear Overhauser Effect) When two nuclear spins are within 5 Å, they will cross-relax. If one spin (S) is saturated (red lines along the edge), the system is not in equilibrium anymore. Magnetization will either flow from the top to the bottom (W2 active) or from the right to left (W0 active). The difference in energy between bb and aa is twice the spectrometer frequency, and molecular motions about that frequency are required for the transition. The difference between ab and ba is very small, and very slow molecular motions (e.g. proteins) will excite that transition. 9/3/2021 94

95. 9/3/2021 Different Types of NMR Electron Spin Resonance (ESR) 1-10 GHz (frequency) used in analyzing free radicals (unpaired electrons) Magnetic Resonance Imaging (MRI) 50-300 MHz (frequency) for diagnostic imaging of soft tissues (water detection) NMR Spectroscopy (MRS) 300-900 MHz (frequency) primarily used for compound ID and characterization

96. 9/3/2021 Explaining NMR UV/Vis spectroscopy Sample

97. 9/3/2021 Explaining NMR

98. 9/3/2021 A Modern NMR Instrument Radio Wave Transceiver

99. Magnet Legs Probe Sample Bore Cryogens Magnet Coil 9/3/2021 NMR Magnet Cross-Section

100. The spectrometer 9/3/2021

101. 9/3/2021

102. 800 MHz 9/3/2021

103. 9/3/2021 NMR Spectroscopy

104. 9/3/2021 The NMR Graph =>

105. 9/3/2021 NMR Samples

106. 9/3/2021 An NMR Probe

107. 9/3/2021 NMR Sample & Probe Coil

108. 9/3/2021 Types of NMR Tubes Solid State Sample Rotors Solution NMR Sample Tube Spinners NMR Sample Tubes with Caps

109. 9/3/2021 NMR Sample Preparation Use clean + dry NMR tubes and caps (tubes can be re-used, caps should not!) 0.5 ml deuterated solvent (i.e. CDCl 3 ,D 2 O , Deuterated acetone etc.) substrate requirements for routine spectra: 10 mg for proton NMR 100 mg for carbon-13 NMR min. filling height of tube: 2 inches (5 cm) Cleaning of tubes: 1. rinse with solvent you were using 2. rinse with acetone 3. dry in (vacuum-)oven at low temperature 5 mm

110. 9/3/2021 NMR Sample Preparation Clean clear solution Suspension or opaque solution Precipitate Not enough solvent Two phases Concentration gradient GOOD! B a d S a m p l e s !

111. 9/3/2021 Good and bad NMR Spectra … are the result of: Sample preparation Choice of solvent Homogeneity of magnetic field Data acquisition parameters Processing procedures

112. 9/3/2021 Good spectrum ppm ppm

113. 9/3/2021 Bad spectrum ?

114. 9/3/2021 Bad spectrum ! Tall signals are cut off

115. 9/3/2021 Bad spectrum ?

116. 9/3/2021 Bad spectrum ! Signals too small (only allowed to compare signal intensities between different spectra)

117. 9/3/2021 Bad spectrum ?

118. 9/3/2021 Bad spectrum ! Broad signals (bad sample, poor shimming, wrong processing parameters)

119. 9/3/2021 Bad spectrum ?

120. 9/3/2021 Bad spectrum ! Signals are distorted (automatic phase correction is often insufficient) Excessive peak picking (low p.p. threshold, also due to improper phasing)

121. 9/3/2021 Applications Determination of exact structure of drugs and drug metabolites - MOST POWERFUL METHOD KNOWN Detection/ quantitation of impurities Analysis/ deconvolution of liquid mixtures

122. 9/3/2021 Analysis of blood, urine and other biofluid mixtures to quantify and identify metabolite changes Allows one to detect drug toxicity and even localize toxicity (for preclinical trials) in a non-invasive way Detection, identification and quantitation of primary and secondary drug metabolites

123. 9/3/2021 Other Applications Clinical testing (detection of inborn errors of metabolism, cancer, diabetes, organic solvent poisoning, drugs of abuse, etc. etc.) Cholesterol and lipoprotein testing Chemical Shift Imaging (MRI + MRS) Pharmaceutical Biotechnology (proteins, protein drugs, SAR by NMR)

124. 9/3/2021 Hydrogen and Carbon Chemical Shifts =>

125. Acknowledgement For lecture notes refer http://redpoll.pharmacy.ualberta.ca http://www.pharmacy.ualberta.ca/pharm325 9/3/2021