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One-Dimensional (1D) NMR Experiments

One-Dimensional (1D) NMR Experiments. 1D NMR - General summary. Relaxation – Preparation – Evolution – Mixing – Acquisition. Relaxation signal fully recovers to +z should be > 5T 1 , normally T 1 to 2T 1 (~1-2 secs.) Preparation select desired information Evolution

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One-Dimensional (1D) NMR Experiments

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  1. One-Dimensional (1D) NMR Experiments 1D NMR - General summary Relaxation – Preparation – Evolution – Mixing – Acquisition • Relaxation • signal fully recovers to +z • should be > 5T1, normally T1 to 2T1 (~1-2 secs.) • Preparation • select desired information • Evolution • related to coupling constant (~1/2J) • Mixing • requires 180 refocusing pulse to phase spectra • usually evolution of through space dipole-dipole relaxation (NOE) • Acquisition • FID is observed usually with decoupling

  2. One-Dimensional (1D) NMR Experiments • Difference Spectroscopy • Determine which signals change between different experiments • vary decoupling frequency • change sample composition (protein-ligand titration) • change delay times (NOE, coupling) • Subtract the two spectra • don’t get perfect cancellation • Instrument instability • Bloch-Siegert shift • Nuclear Overhauser effects Small change in frequency Incomplete cancellation

  3. One-Dimensional (1D) NMR Experiments • Decoupling Difference Spectroscopy • One spectra collected with decoupling off resonance • decoupler set at a frequency far off from any peaks in the spectra • Second spectra collected with selected decoupling of one peak in the spectra • Helps deconvolute complex coupling patterns • repeat for each coupled resonance in the spectra • coupled spectra give positive signals • decoupled spectra give negative signals 1H signals coupled to 31P Difference spectrum (b-a) 1H spectrum with Decoupler set on 31P signal of PPh3 1H Reference spectrum

  4. One-Dimensional (1D) NMR Experiments • Selective Population Transfer • Minimize Bloch-Siegert shift • use weak, selective decoupling pulse • equalizes population of two spin states • effects population of coupled spin states • Changes observed from difference spectra A spins Normal 1:1 A-X doublet 2dN-dN dN-0 0.5:1.5 A-X doublet after selective decoupling 1.5dN-dN 1.5dN-0

  5. NOE 4.1Å 2.9Å One-Dimensional (1D) NMR Experiments • Nuclear Overhauser Effect (NOE) • Dipole-dipole relaxation • through space correlation (<5Å) • stereochemistry and conformation of molecules • Irradiate one nucleus • intensity of nuclei which are close in space change • magnitude change depends on nuclei type • depends on distance between nuclei Relaxation through interaction of spin-states

  6. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Mechanism for Relaxation • Each nuclei creates a magnetic field that effects other nuclei • Dipole-dipole coupling is described by a unit vector that connects the dipoles • head to tail alignment is lowest energy • But structures can constrain relative alignment Field at k created by j Magnetic spins are like bar magnets Magnitude of dipole-dipole interaction may come from numerous interactions

  7. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Important: effect is time-averaged • Gives rise to dipolar relaxation (T1 and T2) and especially to cross-relaxation • Mechanism by which spins return to equilibrium state (aligned with external magnetic field +z) • Will discuss in detail later in the course Perturb 1H spin population affects 13C spin population NOE effect

  8. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE, h) – the change in intensity of an NMR resonance when the transition of another are perturbed, usually by saturation. Saturation – elimination of a population difference between transitions (irradiating one transition with a weak RF field) hi = (I-Io)/Io where Io is thermal equilibrium intensity irradiate N-d bb X A N N ab ba X N+d A aa Observed signals only occur from single-quantum transitions Populations and energy levels of a homonuclear AX system (large chemical shift difference)

  9. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) Saturated (equal population) saturate N-½d bb I S N-½d N+½d ab ba I N+½d S aa Saturated (equal population) Observed signals only occur from single-quantum transitions Populations and energy levels immediately following saturation of the S transitions N-½d bb Relaxation back to equilibrium can occur through: Zero-quantum transitions (W0) Single quantum transitions (W1) Double quantum transitions (W2) W1X W1A N-½d W2 N+½d ab ba W0 W1X W1A aa N+½d The observed NOE will depend on the “rate” of these relaxation pathways

  10. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) N-½d Solomon Equation: bb W1X W1A W2 N-½d N+½d ab ba W0 W1X W1A aa N+½d Steady-state NOE enhancement at spin A is a function of all the relaxation pathways If only W1,noNOE effect at HA If W0 is dominant, decrease in intensity at HA negative NOE If W2 is dominate, increase in intensity at HA positive NOE For homonuclear (gX=gA), maximum enhancement is ~ 50% For heteronuclear (gX=gA), maximum enhancement is ~50%(gX/gA)

  11. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) Intensity of NOE “builds-up” as a function of time (tm – mixing time) • NOE build-up rate is dependent on correlation time (tc) and frequency • correlation time: time it takes a molecule to rotate one radian (360o/2p) • ~10-11 secs. for small molecules • ~10-9 secs. MW:1000 to 3000 • >10-9 secs. MW > 5000

  12. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Correlation Time • Debye theory of electric dispersion: N – viscosity T – temperature a – radius of molecule k – Boltzman constant Varying temperature, viscosity or mass of sample will change tc

  13. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Mechanism for Relaxation • Dipolar coupling between nuclei and solvent (T1) • interaction between nuclear magnetic dipoles • depends on correlation time • oscillating magnetic field due to Brownian motion • depends on orientation of the whole molecule • in solution, rapid motion averages the dipolar interaction –Brownian motion • in crystals, positions arefixed for single molecule, but vary between molecules leading range of frequencies and broad lines. Tumbling of Molecule Creates local Oscillating Magnetic field

  14. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Mechanism for Relaxation • Solvent creates an ensemble of fluctuating magnetic fields • causes random precession of nuclei  dephasing of spins • possibility of energy transfer  matching frequency Field Intensity at any frequency • tc represents the maximum frequency • 10-11s = 1011 rad s-1 = 15920 MHz • All lower frequencies are observed

  15. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) Mechanism for Relaxation Extreme narrowing limit (flat region) tc ~ 10ns (macromolecule) Relaxation or energy transfers only occurs if some frequencies of motion match the frequency of the energy transition. The available frequencies for a molecule undergoing Brownian tumbling depends on tc. The total “power” available for relaxation is the total area under the spectral density function. tc ~ 10ps (small molecule) 1/tc Intensity of fluctuations in magnetic field Proportional to tc (note: different scales)

  16. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Mechanism for Relaxation • Spectral density is constant for w << 1/tc • tc decreases, wo also decreases and T1 increases • at 1/tc≈wo there is a point of inflection • W2 falls off first since it is the sum of two transitions • relaxation rates via dipolar coupling are: NOE is dependent on the distance (1/r6) separating the two dipole coupled nuclei Important: the effect is time-averaged! Extreme narrowing limit: 1/tc >>wo then wo2tc2 <<1)

  17. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Dependence of NOE on tc • NOE can be positive, zero or negative depending on tc MW Zero NOE positive NOE negative NOE Increasing MW Decreasing tc Small molecules Biomolecules, polymers

  18. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • Experimental Aspects of NOE • 50% NOE is theoretically possible • In practice, < 5% NOEs are frequently observed • A number of factors reduces the NOE • Any relaxation pathway other than dipole-dipole will reduce NOE • paramagnetic relaxation most common: paramagnetic transition metal ions or O2  degas sample • viscous, solvents, MW or presence of solvents lower tc lower hmax • NOE builds up by dipole-dipole relaxation • in small molecules, T1DD > 10 secs. To differentiate between NOEs and changes from decoupling, do not decouple during acquisition

  19. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • NOE Difference Spectroscopy • selectively irradiate on resonance • intensity will be perturbed for other spatially close nuclei • subtract spectra with/without irradiation • Aids in the assignment of the NMR spectra Strong NOE must be H3 Irradiate chemically distinct H7

  20. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) • 13C Spectroscopy • nearly always decoupled to enhance signal to noise • lose splitting pattern • intensities are not reliable parameter to quantify number of carbons • different values of NOE • different relaxation times • Quaternary carbons tend to have very long relaxation times and are commonly not observed or severely reduced in intensity • changing when decoupling takes place in pulse sequences can select • between, NOE, 1H coupling and full sensitivity enhancement Decoupling with NOE Decoupling with NOE suppression No 1H decoupling

  21. One-Dimensional (1D) NMR Experiments Nuclear Overhauser Effect (NOE) Decoupling with NOE suppression NOE while maintaining 1H coupled spectra decouple Decoupling with NOE

  22. One-Dimensional (1D) NMR Experiments J Modulation (JMOD) • Used to Edit 13C Spectra • changes the “phase” of C and CH2 signals relative to CH and CH3 • C and CH2 point up (positive) • CH and CH3 point down (negative) • Maximize sensitivity by complete decoupling and NOE, but maintain spin system information. d1 = recycle delay for relaxation d2 = 1/J1H-13C 180o 90o

  23. One-Dimensional (1D) NMR Experiments J Modulation (JMOD) • Aids in NMR Assignments • Identifies the number of different spin systems presents • Chemical shifts identifies the types of functional groups that are present. 10 4 3 1 6 8 7 9 2 5 6 8 2 4 1 5 3 7 9,10

  24. One-Dimensional (1D) NMR Experiments J Modulation (JMOD) On resonance (center of coupling pattern) 180o decouple • Remember Coupling constants are in Hz (cycles per second) • complete cycle is 360o • each spin moves relative to carrier • (center of spin system) during d2 delay • 13C singlet: • on resonance doesn’t move during 1/J • 13CH doublet each spin distance from • carrier is J/2  moves 180o in 1/J • 13CH2 triplet: • - center peak on-resonance doesn’t move. • - outer peaks are J from carrier  moves • 360o in 1/J • 13CH3 quartet: • - inner doublet are J/2 from carrier  • moves 180o in 1/J. Outer • - doublet are 3J/2 from carrier  moves • 540o or an effective 180o in 1/J 13C 13CH 13CH2 13CH3

  25. One-Dimensional (1D) NMR Experiments J Modulation (JMOD) On resonance (center of coupling pattern) 180o decouple • Phase of the Peaks Differ as a result of the Different Spin Systems • the 180o pulse and the second 1/J delay • allows for refocusing of chemicals shifts that • differ from the carrier position • rotation is actually dependent on d+J • 180o reverses direction and refocus rotation due to d • 1J13CH ~ 125-170 Hz • use average J ~ 145 Hz • problems with 13CH of alkynes J ~250 Hz  behaves like 13CH2 • Decoupler is turned on during second d2 and acquisition to collapse spins to singlet and gain NOE sensitivity • If d2 set to 1/2J, only observe 13C • difficult  average J  incomplete cancellation and weak 13C signal 13C 13CH 13CH2 13CH3

  26. One-Dimensional (1D) NMR Experiments INEPT • Polarization Transfer • population difference between a and b states is proportional to g • 1H population difference ~ 4x > 13C • If this difference could be transferred from 1H to 13C, 13C S/N would increase by a factor of 4. • Lose of NOE effect • polarization transfer > NOE effect

  27. One-Dimensional (1D) NMR Experiments INEPT • Polarization Transfer • selective 180o on one 1H spin • inverts the 1H a and b spin states • 13C population differences are now ±DH instead of +DC • Repeat by inverting other 1H spin and subtract spectra  in-phase doublet with 4-fold increase in S/N Selective 180o on H1

  28. One-Dimensional (1D) NMR Experiments INEPT • Polarization Transfer • Previous described experiment is impractical • need to repeat experiment for each unique carbon present in molecule • Can achieve the same effect with the INEPT pulse sequence • simultaneous polarization transfer for all carbons present in molecule • Common module of multidimensional NMR experiments 90o 90o 180o d1 = recycle delay for relaxation d2 = 1/4J1H-13C 180o 90o

  29. One-Dimensional (1D) NMR Experiments INEPT Separation in peaks indicate triplet (J~145Hz) INEPT Pascal Triangle 2J J -1:1 doublet 13CH -1:0:1 triplet 13CH2 -1:-1:1:1 quartet 13CH3

  30. One-Dimensional (1D) NMR Experiments INEPT • Decouple INEPT Experiment • results in selective inversion of one spin in the doublet • same result as selective polarization transfer • during first d2 = 1/4J each spin moves 45o • 180o1H refocusing pulse flips spins (would refocus after another 1/4J delay • 180o X pulse exchanges a and b 1H spins • X attached to a are now attached to b and vice-versa • direction of rotation is reversed • During second d2, each spin moves another 45o and are aligned 180o to each other • 900 X pulse generates X FID with polarization transfer • phase cycling of receiver can alternatively add and subtract spectra Final 1H 90o will place one spin as +z and the other as –z Effectively, a selective 180o on one spin

  31. One-Dimensional (1D) NMR Experiments INEPT • Effect of INEPT Pulse Sequence on 1H spins • because spins are 180o to each other, turning on decoupler will cancel spins  no signal • insert 180o refocusing pulse separated by d3=1/4J delay 180o refocusing pulse X spin state after standard INEPT (p6) Decoupler turned on X collapse to singlet

  32. One-Dimensional (1D) NMR Experiments INEPT • Refocused INEPT can Distinguish CH, CH2 and CH3 • selection of d3 as a function of 1/J determines what spins are observed • only 13C attached to 1H are observed • 0.125/J optimal for all positive signal • 0.25/J only 13CH observed • 0.375/J CH2 are anti-phase (negative) • common component of multidimensional NMR pulse sequences to select desired correlations • INEPT not commonly used to select spin systems  DEPT • INEPT is too sensitive to JXH variations CH

  33. One-Dimensional (1D) NMR Experiments DEPT • Pulse Sequence of Choice to Edit 13C NMR Spectra • not possible to use a simple vector model to explain pulse sequence • involves creating multiple-quantum coherence • variable p3 pulse selects desired spin system and phase • 45o pulse: CH, CH2 and CH3 are all positive • 90o pulse: only CH signal observed • 135o pulse CH and CH3 positive with CH2 being negative • Addition and subtraction of DEPT-45, DEPT-90 and DEPT-135 can generate spectra that • only contains CH, CH2 or CH3 signals 90o ao 180o d1 = recycle delay for relaxation d2 = 1/2J1H-13C

  34. One-Dimensional (1D) NMR Experiments DEPT (DEPT-45 + DEPT-135) – DEPT-90 DEPT-45 - DEPT-135 DEPT-90 Normal Spectra

  35. bb W1X W1A W2 ab ba W0 W1X W1A aa One-Dimensional (1D) NMR Experiments DEPT Wo,W2: multiquantum, forbidden transitions multiple quantum vector does not change during t 13C 90o creates multiple quantum coherence 180o pulse refocus chemical shifts Anti-phase component (amplitude function of sin q) Last 1H pulse Multiquantum component (amplitude function of cos q)

  36. One-Dimensional (1D) NMR Experiments PENDANT • Pulse Sequence of Choice to Edit 13C NMR Spectra • DEPT does not observe non-protonated 13C atoms • PENDANT same sensitivity as DEPT • observes quaternary13C, 13CH, 13CH2 and 13CH3 • quaternary 13C signals are stronger than in JMOD • C/CH2 are opposite phase of CH/CH3 signals • PENDANT with chemical shift information generally sufficient to assign 13C spectrum • ambiguities can be removed with the appropriate DEPT experiment • Only requires collecting one spectrum • pointless to acquire simple 1H decoupled 13C spectrum • replaces JMOD and APT • Again, simple spin vector diagrams are insufficient to describe pulse sequence • Creating multiple quantum coherence 180o 90o d1 = recycle delay for relaxation d2 = 1/4J1H-13C d3 = 5/8J1H-13C

  37. One-Dimensional (1D) NMR Experiments PENDANT • Signals can be Missing from JMOD, INEPT,DEPT or PENDANT • relaxation of peaks occur during delays • worse for broad signals • due to exchange or quadrupolar nucleus

  38. Center peak off-scale 13C satellites One-Dimensional (1D) NMR Experiments INADEQUATE • Detects Carbon-Carbon Coupling • 13C nuclei only 1.08% abundant • weak satellites on either side of strong center peak • probability of two bonded atoms both being 13C is 1.17e-2% • Experiment suppresses strong center peak to detect 13C satellites Identifying 13C-13C connectivity beneficial for NMR assignment of complex molecules.

  39. One-Dimensional (1D) NMR Experiments INADEQUATE • Detects Carbon-Carbon Coupling • delay (d2) can be set to select 1J13C-13C or longer coupling 13C-13C • Two-dimensional version (2D) determines 13C-13C connectivity d1 = recycle delay for relaxation d2 = 1/4J13C-13C 1H decoupling on throughout experiment

  40. One-Dimensional (1D) NMR Experiments INADEQUATE d2 = 0.08 sec J13C-13C = 3 Hz d2 = 0.0062 sec J13C-13C = 40 Hz 13C spectrum

  41. One-Dimensional (1D) NMR Experiments Summary of Information Present in Some 1D Experiments

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