Nuclear Magnetic Resonance Parameters

Transcription

1 Nuclear Magnetic Resonance Parameters P. K. Madhu Department of Chemical Sciences Tata Institute of Fundamental Research omi Bhabha Road Colaba Mumbai , India

2 NMR: What are the Potentials? Understanding the nuclear spin system Spin choreography Characterisation of various materials Structure of compounds, biomolecules Proteins, nucleic acids. Magnetic resonance imaging, MRI

3 Structure elucidation Natural product chemistry Synthetic organic chemistry NMR: What are the Potentials? Study of dynamic processes Reaction kinetics Study of equilibrium (chemical or structural) Structure determination of macromolecules Proteins Nucleotides, protein/dna complexes Polysaccharides Drug Design Structure-Activity Relationship (SAR) by NMR Medicine Magnetic Resonance Imaging Metabonomics: combined use of spectroscopy & multivariate statistical approaches to studies of biofluids, cells & tissues. Gives a unique metabolic fingerprint for each complex biological mixture, sensitive to change

4 NMR Nuclear Magnetic Resonance Nuclear spins are our spies to probe structure Superconducting magnets give us the medium and mechanism to manipulate the spins Talking to the spins in a language that they understand, which means we have to resonate with them

5 Nuclear Spins Nuclear spin is a fundamental property just like charge and mass Electron Proton Neutron Deuterium atom: 1 electron, 1 proton, and 1 neutron Electronic spin = 1/2 Nuclear spin = 1 NMR uses the nuclear properties and nuclear spins to eavesdrop on others Paired nuclear spins are or of no use to work as nuclear spies NMR needs unpaired, socially non-committed, nuclear spins to act as spies elium atom Net nuclear spin = 0 NMR non-observabe

6 NMR Periodic Table: Nuclei with Spins-1/2 n X element with one I=1/2 isotope 1 2 Li 11 Na 19 K 37 Rb 55 Cs 87 Fr 3 Be 12 Mg 20 Ca Sr Ba 88 Ra 21 Sc 39 Y La Ac 22 Ti 40 Zr 72 f 23 V 41 Nb 73 n X Ta 24 Cr element with two (three) I=1/2 isotopes Mo W Mn Tc 75 Re Fe Ru Os Co Rh Ir Ni Pd 78 Pt Cu Ag 79 Au Zn Cd g B 13 Al 81 Ga In C Si Ge 50 Sn N P As Sb O S Se Te 9 35 F 17 Cl 53 Br I 2 e 10 Ne 18 Ar 36 Kr 54 Xe Tl Pb Bi Po At Rn Common nuclei probed are 13 C, 15 N, 29 Si, and 31 P

7 NMR Periodic Table: Nuclei with alf-integer Spins>1/2 1 2 Li 11 Na 19 K 37 Rb 55 Cs 87 Fr 3 Be 12 Mg 20 Ca Sr Ba 88 Ra 21 Sc 39 Y La Ac 22 Ti 40 Zr 72 f 23 V 41 Nb 73 n X Ta 24 Cr Mo W n X Mn Tc 75 Re Fe Ru Os Co Rh Ir X I = 3/2 I = 5/2 I = 7/2 n X n Ni Pd 78 Pt n X I = 9/2 5/2 + 7/2 etc. Cu Ag 79 Au Zn Cd g B 13 Al 81 Ga In C Si Ge 50 Sn N P As Sb O S Se Te 9 35 F 17 Cl 53 Br I 2 e 10 Ne 18 Ar 36 Kr 54 Xe Tl Pb Bi Po At Rn Many of the nuclei of industrial importance are quadrupolar spin nuclei manifesting in ceramics, glasses, zeolites and catalysts

8 Nuclear Spins Charge Nuclear spin Spin μ = γ I Magnetic moment Gyromagnetic ratio, depends on the nucleus Spin quantum number

9 Nuclear Spins & Magnetic Field A spinning gyroscope in a gravity field A spinning charge in a magnetic field

10 Nuclear Spin

11 NMR Spies Inside a Magnetic Field

12 NMR Spies In Action

13 NMR Spies The Tools ow do the spins probe the medium? Chemical shift anisotropy Dipole-dipole couplings Through-bond couplings Quadrupolar couplings

14 Nuclear Spin Interactions Spin Interactions External Internal Zeeman, Z RF, RF (t) CSA, CS Dipole, DD Scalar, J Quad, Q The isotropic parts (manifest in solution-state) are time independent Anisotropic parts cause line broadening

15 Internal Nuclear Spin Interactions Spin>½, 23 Na, 17 O.. Spin Interactions Spin ½, 1, 13 C.. Electric Magnetic Quadrupolar Chemical shift Spin-spin couplings Isotropic chemical shift Chemical shift anisotropy, CSA Scalar, J- couplings Dipolar Isotropic quad. shift 1 st, 2 nd order quad. Interaction, anisotropic eteronuclear omonuclear

16 NMR: Nuclear Spins, Magnetic Moments, and Resonance

17 The Electromagnetic Spectrum

18 B 0, External magnetic field RF waves in Nuclear magnetic moments in the sample The frequency of emitted RF waves reveals information about the magnetic environment of atomic nuclei RF waves out

19 Populations and Nuclear Magnetisation

20 Nuclear Magnetic Resonance

21 C 3 -C 2 -O Typical 1 NMR Spectrum

22 Sensitivity of NMR Spectroscopy

23 Sensitivity of NMR Spectroscopy Isotope Net Spin γ / Mz T -1 Abundance % 1 1/ / P 1/ Na 3/ N N 1/ C 1/ F 1/ Relative sensitivity of nuclei depends on Gyromagnetic ratio (γ)( Natural abundance of the isotope

24 Sensitivity of NMR Spectroscopy 1 NMR spectra of organic compound 8 scans ~12 secs 13 C NMR spectra 8 scans ~12 secs 13 C NMR spectra 10,000 scans ~4.2 hours

25 Energy Levels: Why Big Magnets Are Needed?

26 Do We Need Bigger/igher Magnets? 30 Mz 7 kgauss 500 Mz 117 kgauss ighest superconducting magnet currently available is 23 T yielding proton Larmor frequency of 1000 Mz

27 Chemical Shift- Usefulness of NMR NMR used for structural characterisation Should be able to distinguish functional groups unambiguously C 3 C 2 O

28 Chemical Shift- Usefulness of NMR b C a c Cl ν a ν b ν c frequency

29 Pioneer of NMR in India Prof. Dharmatti was a student of Felix Bloch in Stanford and discovered chemical-shift phenomenon

30 Fourier-Transform NMR

31 Typical Experiment in NMR: RF Pulse z M o z z y y M o y x x x RF on RF off RF off Effect of a 90 o x pulse

32 After the Pulse: Nuclear Spin Evolution z z z M o y y y x x M o ω x RF receivers pick up the signals I x Time y The spins precess in the xy plane and relax to the equilibrium value, free induction decay

33 Fourier Transformation

34 Radio-Frequency Pulse 90 x Amplitude Volume Pulse Phase Music Timing Frequency Pitch

35 Spin Relaxtion There are two primary causes of spin relaxation: Spin - lattice relaxation, T 1, longitudinal relaxation lattice Spin - spin relaxation, T 2, transverse relaxation

36 Spin Relaxation, T 1 T 1 determines the repetition rate of an experiment 90 x Signal-to-noise enhancement n For an optimum signal, there is a need to wait for a few T 1 times before which an experiment can be repeated for signal averaging The time scale with which the z-component of the magnetisation has felaxed back to the equilibrium magnetisation, M 0

37 Behaviour of T 1 and T 2 Relaxation Times Long Gases T 1 and T 2 at short correlation times Small molecules Large molecules T 1 T 1 or T 2 relaxation time Medium sized molecules T 1 minimum Short T ns Correlation time Fast motion Short τ c Slow motion Long τ c optimal frequency for T 1 relaxation (Mz frequencies)

38 T 1 Measurement- Inversion Recovery Experiment 180 x 90 x τ z z τ=0 y 90 x y x z x z some τ y 90 x y x z x z large τ y 90 x y x x

39 T 1 Measurement- Inversion Recovery Experiment 180 x 90 x τ t Measure the NMR signal as a function of τ 0 τ dm z M M = dt T Bloch equation for longitudinal magnetisation: z 0 M = M e z 1 τ (1 2 T1 ) 0

40 T 1 Measurement- Inversion Recovery Experiment Inversion Recovery - Measure NMR Intensity as a function of the delay time τ and fit to an exponential function 0 τ M z M z = M o (1-2e -τ/t1 ) 0 τ

41 Transverse Relaxation Time, T 2 Current amplitude time frequency The faster the dephasing, the faster the decay of the time domain signal, the broader the line Line widths are related to T 2 relaxation. LW ~ 1/ T 2 (not always true due to inhomogeneous broadening) T 2 is always faster (shorter) than or equal to T 1

42 T 2 Measurement- Spin-Echo Experiment 90 x 180 x τ/2 τ/2 t Bloch equation for transverse magnetisation: M x (t) dm dt M M xy, xy, = = Me xy, 0 T 1 τ T 2 τ Plot of the peak amplitude as a function of τ in the spin-echo experiment

43 Spin-Echo Experiment Physics Today, 1953 E. ahn, Physical Reivew, 80, 1950

44 Spin-Echo Experiment 90 x 180 x τ/2 τ/2 t z x x 90 x evolution y y y 180 x x x Magnetisation is refocused Formation of an echo Decay of echo only due to T 2 y evolution y

45 T 2 Relaxation, Line Width and Correlation Times τ c = 4pη w r 3 3k B T η w = viscosity of the solvent r 3 = hydrated radius 25 Δν FWM (z) τ c (ns) See Cavanagh et al. Protein NMR spectroscopy, pages

46 T 2 Relaxation, Line Width and Correlation Times mobile, flexible chain has narrower line widths than globular protein N N N N N 15 N

47 T 1 Relaxation and Mobility Mobility is also expressed in T 1 relaxation times. N N N N N t = 10 us t = 100 us t = 1000 us t = 5000 us

48 Intensity and Resolution To summarise: The first point of the FID determines the intensity of the resonance signal The duration of the FID determines the resolution of the resonance signal

49 Information in a NMR Spectra 1) Energy E = hυ γ-rays x-rays UV VIS IR μ-wave radio h is Planck constant υ is NMR resonance frequency wavelength (cm) Observable Name Quantitative Information Peak position Chemical shifts (δ) δ(ppm) = u obs u ref /u ref (z) chemical (electronic) environment of nucleus Peak Splitting Coupling Constant (J) z peak separation neighboring nuclei (intensity ratios) (torsion angles) Peak Intensity Integral unit less (ratio) nuclear count (ratio) relative height of integral curve quantifying Peak Shape Line width Δυ = 1/πT 2 molecular motion peak half-height chemical exchange

50 Chemical Shift a) Small local magnetic fields (B loc ) are generated by electrons as they circulate around nuclei. b) These local magnetic fields can either oppose or augment the external magnetic field 1) Typically oppose external magnetic field 2) Nuclei see an effective magnetic field (B eff ) smaller then the external field 3) σ magnetic shielding or screening constant i. depends on electron density ii. depends on the structure of the compound B eff = B o -B loc B eff = B o ( 1 - σ ) O-C 2 -C 3 σ reason why we observe three distinct NMR peaks instead of one based on strength of B 0 ν = γb o /2π de-shielding high shielding Shielding local field opposes B o

51 Proton Peaks are Referenced Against TMS Rather than measure the exact resonance position of a peak, we measure how far downfield it is shifted from TMS. C 3 C 3 Si C 3 C 3 tetramethylsilane TMS ighly shielded protons appear way upfield. n shift in z downfield TMS 0

52 igher Fields (Frequencies) Give Larger Shifts The shift observed for a given proton in z also depends on the frequency of the instrument used. igher frequencies = larger shifts in z n shift in z downfield TMS 0

53 ppm and z 1 operating frequency z equivalent of ppm 500 Mz 500 z 600 Mz 600 z 800 Mz 800 z

54 Chemical Shift parts per million chemical shift = δ = shift in z spectrometer frequency in Mz = ppm igher frequency leads to more dispersion 500 Mz 10 PPM 600 Mz 0 10 PPM 800 Mz 0 10 PPM 0

55 Chemical-Shift Scales For protons, ~ 15 ppm: Acids Aldehydes Aromatics Amides Olefins Alcohols, protons a to ketones Aliphatic ppm C For carbon, ~ 220 ppm: C=O in ketones Aromatics, conjugated alkenes Olefins 2 0 TMS Aliphatic C 3, C 2, C 3 C Si C 3 C 3 ppm TMS C=O of Acids, aldehydes, esters Carbons adjacent to alcohols, ketones

56 Chemical-Shift Scales acid COO aldehyde CO benzene C alkene =C- C- where C is attached to an electronegative atom X-C- 4 C on C next to pi bonds X=C-C- aliphatic C Interpretation of most 1D spectra is possible with this information

57 Factors Determining Resonance Positions of 1 Deshielding by electronegative elements Anisotropic fields usually due to pi-bonded electrons in the molecule Deshielding due to hydrogen bonding

58 Deshielding by an Electronegative Element δ- δ+ Cl electronegative element C δ- δ+ Chlorine deshields the proton, that is, it takes valence electron density away from carbon, which in turn takes more density from hydrogen deshielding the proton. deshielded protons appear at low field highly shielded protons appear at high field deshielding moves proton resonance to lower field 0 ppm

59 Electronegativity Dependence of Chemical Shift Dependence of the Chemical Shift of C 3 X on the element X Compound C 3 X C 3 F C 3 O C 3 Cl C 3 Br C 3 I C 4 (C 3 ) 4 Si Element X F O Cl Br I Si Electronegativity of X Chemical shift δ most deshielded deshielding increases with the electronegativity of atom X TMS

60 Substitution Effects on Chemical Shift most deshielded CCl 3 C 2 Cl 2 C 3 Cl ppm The effect increases with greater numbers of electronegative atoms most deshielded -C 2 -Br -C 2 -C 2 Br -C 2 -C 2 C 2 Br ppm The effect decreases with increasing distance

61 Aromatic Ring Current Effects The presence of pi-bonds leads to anisotropic fields and affect chemical shift Aromatic ring currents are observed in molecules like benzene and naphthalene The applied magnetic field gives rise to a ring current in the delocalised pi-electrons of the aromatic ring Benzene rings have the greatest effect Ring protons can get deshielded (induced and static fields add) while inner protons get shielded (induced and static fields oppose) Ring current Induced field

62 Aromatic Ring Current Effects Ring Current in Benzene Ring protons come at 7.3 ppm instead of the 5.6 ppm of the vinylic protons in cyclohexane Circulating π electrons Deshielded fields add together B o Secondary magnetic field generated by circulating π electrons deshields aromatic protons

63 Influence of ydrogen Bonding R O C O O C O R Carboxylic acids have strong hydrogen bonding - they form dimers. With carboxylic acids the O- absorptions are found between 10 and 12 ppm very far downfield. 3 C O O O In methyl salicylate, which has strong internal hydrogen bonding, the NMR absortion for O- is at about 14 ppm, way, way downfield.

64 ydrogen Bond and Chemical Shift R O O R O R The chemical shift depends on how much hydrogen bonding is taking place. Alcohols vary in chemical shift from 0.5 ppm (free O) to about 5.0 ppm (lots of bonding). ydrogen bonding lengthens the O- bond and reduces the valence electron density around the proton - it is deshielded and shifted downfield in the NMR spectrum.

65 Influence of ydrogen Bonding free -bonded O upfield shifted, No -bonding O downfieldfield shifted, -bonding

66 SPIN-SPIN SPLITTING

67 Multiplets in Spectra Splitting of the resonances, multiplets, are due to spin-spin coupling and Can be predicted by the n+1 rule

68 Scalar Coupling e - I 2 I e - 1 Fermi contact J-coupling is facilitated by the electrons in the bonds that connect the nuclei Scalar coupling, through-bond coupling The coupling constants can be related to a number of physical properties: ybridisation, dihedral bond angles, and electronegativity of substituents Geminal and vicinal couplings may be used to determine the bond angles and torsion angles

69 Geminal Scalar Coupling: 2 J Coupling Smaller the J value, larger the bond angle

70 Vicinal Scalar Coupling: 3 J Coupling The magnitude of the J coupling is dictated by the torsion angle between the two coupling nuclei according to the Karplus equation. θ J = A + B cos(θ) + C cos 2 (θ) C C

71 right-handed alpha helix 3 J Nα = 3.9 antiparallel beta sheet 3 J Nα = 8.9 parallel beta sheet 3 J Nα = 9.7 Scalar Coupling

72 Spin System Classification Chemical equivalence among the spins: If the spins of the same isotopic species There exists a molecular symmetry operation that exchanges the two spins a Fa 1,1-difluoroethene Magnetic equivalence among the spins: b Fb This is a stronger form of the chemical equivalence The spins should have the same chemical shifts Either the spins have identical couplings to all other spins in the molecule or there are no other spins in the molecule a 1 b a 3 b a 2 b C 3 -C 2 -OR 3 OR 2 2 OR 1 1 OR 3

73 Spin System Classification a b Fa Fb Not magnetically equivalent δ a = δ b, and δ Fa = δ Fb but J afb J afa and J bfb J bfa Also here J a J b 0 It is an AA XX spin system a 1 b a 3 b a 2 b Magnetically equivalent 3 OR 2 2 OR 1 1 OR 3 It is an A 2 X 3 spin system The most shielded spin is notated as A F F In this case, difluoromethane, 1 s and 19 Fs are magnetically equivalent not due to rotation, but to symmetry around the carbon. It is an A 2 X 2 system

74 Scalar Coupling and Splitting of Resonances this hydrogen peak is split by its two neighbours these hydrogens are split by their single neighbour C C C C two neighbours n+1 = 3 triplet one neighbour n+1 = 2 doublet n+1 rule

75 Exception To The n+1 Rule 1) Protons that are equivalent by symmetry usually do not split one another X C C Y X C 2 C 2 Y no splitting if x=y no splitting if x=y 2) Protons in the same group usually do not split one another C or C

76 Some Splitting Patterns X C C Y C 3 C ( x = y ) C 2 C C 3 C 2 X C 2 C 2 ( x = y ) Y C 3 C 3 C

77 Coupling Constant J C C J J J J J The coupling constant is the distance J (measured in z) between the peaks in a multiplet. J is a measure of the amount of interaction between the two sets of hydrogens creating the multiplet.

78 Representative Coupling Constants vicinal trans C C C C 6 to 8 z 11 to 18 z three bond three bond 3 J 3 J cis geminal C C C 6 to 15 z 0 to 5 z three bond two bond 3 J 2 J

79 Scalar Coupling- Splitting of Resonances J = 0 J 0 I S I S A splitting of a signal means that we have more energies involved in the transition of a certain nuclei. So why do we have more energies? Coupling constants do not depend on the applied magnetic field, unlike the CSA

80 Scalar Coupling- Splitting of Resonances 19 F 1 Nucleus B o Electron 19 F 1 The nuclear magnetic moment of 19 F polarises the F bonding electron (up), which, since we are following quantum mechanics rules, makes the other electron point down (the electron spins have to be antiparallel). Now, since we have different states for the 1 electrons depending on the state of the 19 F nucleus, we will have slightly different energies for the 1 nuclear magnetic moment (remember that the 1s electron of the 1 generates an induced field ). This difference in energies for the 1 result in a splitting of the 1 resonance line.

81 Scalar Coupling- Splitting of Resonances 1 1 B o C Similar analysis for a C 2 system as well. The state of one of the 1 spins gets transmitted to the other 1 spins via the electrons in the bond (things get a bit complicated here due to the sp 3 hybridiation etc. in general).

82 Shift of A is Affected by the Spin of its Neighbour aligned with B o opposed to B o 50 % of +1/2-1/2 molecules A C C C C A 50 % of molecules B o downfield neighbour aligned upfield neighbour opposed At any given time about half of the molecules in solution will have spin +1/2 and the other half will have spin -1/2.

83 Spin Arrangements one neighbour n+1 = 2 doublet one neighbour n+1 = 2 doublet C C C C yellow spins blue spins The resonance positions (splitting) of a given hydrogen is affected by the possible spins of its neighbor.

84 Spin Arrangements two neighbors n+1 = 3 triplet one neighbor n+1 = 2 doublet C C C C methylene spins methine spins

85 Scalar Coupling- Energy Level Picture A X J = 0 J > 0 A X J = 0 A 2 B o E 4 E 3 E 2 A 2 A 2 E A 1 ν A 1 = ν A 2 E 1 A 1 A 1 J > 0 A 1 A 2 E 4 A X J = 0 J < 0 A X ν A 1 ν A 2 B o E 3 E 2 A 2 A 2 E J < 0 A 2 A 1 A 1 A 1 E 1 ν A 2 ν A 1

86 Scalar Coupling: First-Order Spectra In weakly coupled spin systems: Consider ethyl acetate, C 2 at 4.5 ppm and C 3 at 1.5 ppm J is typically 7 z, hence weakly coupled, also called first-order spin system ββ αβ βα αα βββ ααβ αβα βαα αββ βαβ ββα ααα C 2 C 3 Each 1 in C 3 will see three possible states of 1 in C 2 Each 1 in C 2 will see four possible states of 1 in C 3 Note the 1 in C 3 and that in C 2 are equivalent

87 Scalar Coupling: First-Order Spectra 1 st order systems (continued) A coupled to n identical nuclei X (of spin 1 / 2 ) yields n + 1 lines in the spectrum of A. Therefore, the C 2 in EtOAc will show up as four lines, or a quartet. Analogously, the C 3 in EtOAc will show up as three lines, or a triplet. The separation of the lines will be equal to the coupling constant between the two types of nuclei (C 2 s and C 3 s in EtOAc, approximately 7 z). Intensities can also be derived from the diagram of the possible states: Since we have the same probability of finding the system in any of the states, and states in the same rows have equal energy, the intensity will have a ratio 1:2:1 for the C 3, and a ratio of 1:3:3:1 for the C 2. J (z) C 3 C ppm 1.5 ppm

88 Scalar Coupling: First-Order Spectra The splitting of the resonance of a nuclei A by a nuclei X with spin number I will be 2I + 1. Start with one 1 8 Coupling to the first 1 (2 * 1 / = 2) 4 4 In general, the number of lines in these cases will be a binomial expansion, known as the Pascal Triangle: Coupling to the second Coupling to the third : n / 1 : n ( n - 1 ) / 2 : n ( n - 1 ) ( n - 2 ) / 6 :...

89 Scalar Coupling: First-Order Spectra ere n is the number of equivalent spins 1/2 we are coupled to: The results for several n s is In a spin system in which we have a certain nuclei coupled to more than one nuclei, all first order, the splitting will be basically an extension of what we saw before. 7 z Say that we have a C (A) coupled to a C 3 (M) with a J AM of 7 z, and to a C 2 (X) with a J AX of 5 z. We basically go in steps. First the big coupling, which will give a quartet: 5 z Then the small coupling, which will split each line in the quartet into a triplet: This is called a triplets of quartet (big effect is the last ).

90 2-Nitropropane C 3 C C 3 O N + O - 1:6:15:20:16:6:1 in higher multiplets the outer peaks are often nearly lost in the baseline

91 Scalar Coupling: Strong Coupling ω ω πj 0A 0X AX A 2 J > 0 ββ 4 Second-order order spectra, AB spin system B 2 αβsinθ+βαcosθ αβcosθ+βαsinθ 3 B 1 1 αα A sin 2θ 1+sin 2θ J AB 1+sin 2θ J AB 1-sin 2θ ω 0A ω 0X E 4 = / 2 / ω 2 0A + 0A 1 1 / 2 / ω 2 0BX + 0BX 1 1 / 4 / πj 4 πj AB AB E 3 = / 2 / D- 2 D- 1 1 / 4 / πj 4 πj AB AB E 2 = / 2 / D / 4 / πj 4 πj AB AB E 1 = / 2 / ω 2 0A - 0A / 2 / ω 2 0B + 0B 1 1 / 4 / πj 4 πj AB AB sin sin 2θ=J/D 2θ=J/D sos2θ= (ω (ω 0A - 0A / 2 / ω 2 0BX )/D 0BX )/D D=[(ω D=[(ω 0A - 0A -ω 0B ) 2 0B ) 2 +πj +πj 2 2 ]] 1/2 1/2

92 Scalar Coupling: Second-Order Spectra As Δν approaches J, more and more transition of similar energy occur Δν >> J Our system is now a second-order system. We have effects that are not predicted by the simple multiplicity rules that were described earlier Δν = 0

93 Scalar Coupling: Second-Order Spectra AB system has the roofing effect: coupled pairs will lean towards each other, making a little roof: The chemical shifts of nuclei A and B are not at the center of the doublets. They will be at the center of mass of both lines. Δν the ν A - ν B chemical shift difference A ν A ν Z ν B B Δν Δν 2 2 = (( f 1 f -f 1 -f 4 ) 4 )(( f 2 f -f 2 -f 3 ) 3 ) ν A = A ν Z - Z -Δν // 2 ν B = B ν Z + Z Δν Δν // 2 Peak intensities can be computed similarly: J AB AB = f 1 f -f 1 -f 2 2 = f 3 f -f 3 -f 4 4 I 2 I I 2 3 I 3 f 1 f -f 1 -f 4 4 = = I 1 I I 1 4 I 4 f 2 f -f 2 -f 3 3

94 Scalar Coupling: Second-Order Spectra Cl X Cl Cl Cl A Cl Cl A Cl Cl A B A Transition from A 2 X to A 2 B

95 J and Magnetic Field: Comparison Coupling constants are constant - they do not change at different field strengths J = 7.5 z 200 z 7.5 z 100 z 100 Mz Mz Separation is larger 400 z 200 z The shift is dependant on the field J = 7.5 z 7.5 z ppm

96 J and Magnetic Field: Comparison 100 Mz J = 7.5 z 200 z J = 7.5 z 100 z Mz Note the compression of multiplets in the 200 Mz spectrum when it is plotted on the same scale as the 100 Mz spectrum instead of on a chart which is twice as wide. Separation is larger J = 7.5 z 400 z 200 z ppm

97 3 2 1 J and Magnetic Field: Comparison Why buy a higher field instrument? 50 Mz J = 7.5 z Spectra are simplified! Mz 2 1 J = 7.5 z Overlapping multiplets are separated Mz Second-order effects are minimized. J = 7.5 z

98 INTEGRATION

99 Integration of a Peak Besides identifying the type of hydrogen, we can also obtain the relative Numbers of each type of hydrogen by integration Integration is determining the area under a peak The are under a peak is proportional to the number of hydrogens that generate the peak

100 55: 22: 33 = 5: 2: 3 simplest ratio of the heights Integration of a Peak The integral line rises an amount proportional to the number of in each peak Benzyl Acetate integral line

101 Summary So Far! Each different type of hydrogen gives a peak or group of peaks (multiplet) The chemical shift, in ppm, is suggestive of the type of hydrogen generating the peak The integral gives the relative numbers of each type of hydrogen Spin-spin splitting gives the number of hydrogens on adjacent carbons The coupling constant J also gives information about the arrangement of the atoms involved, dihedral angles for instance

102 13 C NMR 12 C is not NMR active, I=0 13 C is NMR active, I=1/2 owever, 13 C is only 1.08% abundant: Low gyromagnetic ratio Signals about 6000 times weaker than 1 The chemical-shift range of 13 C is larger than that of 1, about ppm

103 13 C NMR For a given field strength 13 C has its resonance at a different (lower) frequency than 1. 1 Divide the hydrogen frequency by 4 (approximately) for carbon T 500 Mz 14.1 T 600 Mz T 800 Mz 13 C 11.7 T 125 Mz 14.1 T 150 Mz T 200 Mz

104 13 C NMR Due to the low natural abundance of 13 C spins, the probability of finding two 13 C atoms next to each other in a single molecule is very small 13 C-!3 C coupling NO! Not probable 13 C spectra are determined by many molecules contributing to the spectrum, each having only one 13 C atom owever, 13 C does couple to 1 (spin ½) 13 C- 1 coupling YES! Very common

105 13 C- 1 J Coupling, eteronuclear Coupling 3 protons 2 protons 1 proton 0 protons 13 C 13 C 13 C 13 C n+1 = 4 n+1 = 3 n+1 = 2 n+1 = 1 Methyl carbon Methylene carbon Methine carbon Quaternary carbon The effect of attached protons on 13 C resonances ( n+1 rule applies ) (J s are large ~ z)

106 13 C- 1 J Coupling, eteronuclear Coupling Ethyl phenylacetate 13 C coupled to the hydrogens

107 Decoupling: Removing J Couplings 13 C NMR spectrum of quinoline 1 decoupled 13 C spectrum 13 C spectrum scalar coupled to 1 In cases of many nuclear spins, J couplings can reduce the sensitivity snd crowd the spectra, hence, the need to remove them- eteronuclear/homonuclear J decoupling

108 eteronuclear Decoupling in Solution-State NMR 1 I Decoupling 13 C S (π/2) y S spin detection RF Decoupling

109 eteronuclear Decoupling in Solution-State NMR

110 Double-Resonance Techniques eteronuclear decoupling- A double-resonance scheme Others being Nuclear Overhauser Effect, Spin Tickling and other sophisticated schemes NMR Spectroscopy, A Physicochemical View- Robin arris Modern NMR Techniques for Chemistry Research- Andrew Derome NMR: The Toolkit- P. J. ore, J. Jones, and S. Wimperis Spin Dynamics, Basics of NMR- Malcolm. Levitt

111 Decoupling: Spectral Simplification Ethyl phenylacetate

112 13 C Chemical Shift Chart R-C 3 RANGE 8-30 Saturated carbon - sp 3 no electronegativity effects R-C 2 -R R 3 C / R 4 C Saturated carbon - sp 3 electronegativity effects C-O C-Cl C-Br Unsaturated carbon - sp 2 C=C C C Alkyne carbons - sp Aromatic ring carbons C=O Acids Amides Esters Anhydrides C=O Aldehydes Ketones

113 13 C Chemical Shift Chart nitriles acid anhydrides acid chlorides amides esters carboxylic acids aldehydes α,β-unsaturated ketones ketones ppm 13 C PPM Chart for Carbonyl and Nitrile Functional Groups

114 Structural Determination with 1D NMR Structure determination of small molecules possible with 1D NMR Chemical shift, J coupling, geometry.. Other sophisticated experiments INEPT DEPT Nuclear Overhauser Effect, NOE Relaxation measurements Exchange experiments Diffusion experiments Variable temperature experiments

115 omodecoupling Simple & quick means of determining if two spins are coupled Effective on molecules with simple, well-dispersed spectra Involves irradiation of selected resonance with low power, thus eliminating any coupling to this spin Comparison of homodecoupled spectrum with the normal one, coupling partners of the irradiated peak are determined Normal spectrum C 2 -C 3 Irr. at quartet 2D counterpart Irr. at triplet COSY omodec. spectrum of ethyl benzene

116 Conclusions NMR is a very powerful method to probe geometry, dynamics, and other structural information parameters Chemical shift, scalar coupling, relaxation rate constants, peak integrals are some of the methods used in one-dimensional NMR 1D NMR can yield a host of information regarding a wide range of molecules 1 and 13 C are the normal probes in 1D NMR Other probes, such as, 31 P, 29 Si, 11 B, and many other spin ½ or spin higher nuclei may be used to characterise materials NMR also is a thorough test bed for many quantum mechanics principles A versatile tool with far reaching implications in Physics, Chemistry, Biology, and Medicine