OneDimensional 1 D NMR Experiments 1 D NMR

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

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

One-Dimensional (1 D) 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 Ø repeat for each coupled resonance in the spectra coupled spectra give positive signals Ø decoupled spectra give negative signals 1 H Difference spectrum (b-a) 1 H spectrum with Decoupler set on 31 P signal of PPh 3 1 H Reference spectrum signals coupled to 31 P

One-Dimensional (1 D) NMR Experiments Selective Population Transfer -Minimize Bloch-Siegert shift use weak, selective decoupling pulse Ø equalizes population of two spin states Ø effects population of coupled spin states Changes observed from difference spectra Ø A spins Normal 1: 1 A-X doublet 2 d. N-d. N-0 0. 5: 1. 5 A-X doublet after selective decoupling 1. 5 d. N-d. N 1. 5 d. N-0

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) -Dipole-dipole relaxation -through space correlation (<5Å) Ø stereochemistry and conformation of molecules NOE 4. 1Å 2. 9Å Irradiate one nucleus Ø intensity of nuclei which are close in space change Ø magnitude change depends on nuclei type Ø depends on distance between nuclei Relaxation through interaction of spin-states

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

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) a) b) Important: effect is time-averaged Gives rise to dipolar relaxation (T 1 and T 2) and especially to crossrelaxation Ø Mechanism by which spins return to equilibrium state (aligned with external magnetic field +z) Ø Will discuss in detail later in the course Perturb 1 H spin population affects 13 C spin population NOE effect

One-Dimensional (1 D) 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. hi = (I-Io)/Io where Io is thermal equilibrium intensity Saturation – elimination of a population difference between transitions (irradiating one transition with a weak RF field) irradiate ab N A bb N-d X ba X aa N+d N A Populations and energy levels of a homonuclear AX system (large chemical shift difference) Observed signals only occur from single quantum transitions

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) Saturated (equal population) ab N-½d S bb N-½d saturate I ba I aa N+½d S Saturated (equal population) Observed signals only occur from single quantum transitions Populations and energy levels immediately following saturation of the S transitions ab W N-½d 1 A W 1 X bb N-½d W 2 W 0 aa N+½d W 1 X N+½d ba W 1 A Relaxation back to equilibrium can occur through: Zero quantum transitions (W 0) Single quantum transitions (W 1) Double quantum transitions (W 2) The observed NOE will depend on the “rate” of these relaxation pathways

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) ab W 1 A N-½d W 1 X bb N-½d W 2 W 0 aa N+½d Solomon Equation: W 1 X N+½d ba W 1 A Steady-state NOE enhancement at spin A is a function of all the relaxation pathways If only W 1, no NOE effect at HA If W 0 is dominant, decrease in intensity at HA negative NOE If W 2 is dominate, increase in intensity at HA positive NOE For homonuclear (g. X=g. A), maximum enhancement is ~ 50% For heteronuclear (g. X=g. A), maximum enhancement is ~50%(g. X/g. A)

One-Dimensional (1 D) 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 (360 o/2 p) – ~10 11 secs. for small molecules –~10 9 secs. MW: 1000 to 3000 –>10 9 secs. MW > 5000

One-Dimensional (1 D) 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

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

One-Dimensional (1 D) 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 t possibility of energy transfer matching frequency t Field Intensity at any frequency tc represents the maximum frequency – 10 -11 s = 1011 rad s-1 = 15920 MHz All lower frequencies are observed

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) Mechanism for Relaxation Extreme narrowing limit (flat region) tc ~ 10 ns (macromolecule) tc ~ 10 ps (small molecule) 1/tc Intensity of fluctuations in magnetic field Proportional to tc (note: different scales) 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.

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) Mechanism for Relaxation • Spectral density is constant for w << 1/tc tc decreases, wo also decreases and T 1 increases t at 1/tc ≈ wo there is a point of inflection – W 2 falls off first since it is the sum of two transitions t relaxation rates via dipolar coupling are: t NOE is dependent on the distance (1/r 6) separating the two dipole coupled nuclei Important: the effect is time-averaged! Extreme narrowing limit: 1/tc >>wo then wo 2 tc 2 <<1)

One-Dimensional (1 D) 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 Small molecules Increasing MW Decreasing tc Biomolecules, polymers

One-Dimensional (1 D) 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 t Any relaxation pathway other than dipole will reduce NOE – paramagnetic relaxation most common: paramagnetic transition metal ions or O 2 degas sample t viscous, solvents, MW or presence of solvents lower tc lower hmax t NOE builds up by dipole relaxation in small molecules, T 1 DD > 10 secs. – To differentiate between NOEs and changes from decoupling, do not decouple during acquisition

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) NOE Difference Spectroscopy • selectively irradiate on resonance intensity will be perturbed for other spatially close nuclei t subtract spectra with/without irradiation • Aids in the assignment of the NMR spectra t Strong NOE must be H 3 Irradiate chemically distinct H 7

One-Dimensional (1 D) NMR Experiments Nuclear Overhauser Effect (NOE) 13 C Spectroscopy • nearly always decoupled to enhance signal to noise lose splitting pattern t intensities are not reliable parameter to quantify number of carbons Ø different values of NOE Ø different relaxation times t – 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, 1 H coupling and full sensitivity enhancement Decoupling with NOE suppression No 1 H decoupling

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

One-Dimensional (1 D) NMR Experiments J Modulation (JMOD) Used to Edit 13 C Spectra • changes the “phase” of C and CH 2 signals relative to CH and CH 3 C and CH 2 point up (positive) t CH and CH 3 point down (negative) • Maximize sensitivity by complete decoupling and NOE, but maintain spin system information. t d 1 = recycle delay for relaxation d 2 = 1/J 1 H-13 C 90 o 180 o

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

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

One-Dimensional (1 D) NMR Experiments J Modulation (JMOD) Phase of the Peaks Differ as a result of the Different Spin Systems On resonance (center of coupling pattern) 13 C • the 180 o pulse and the second 1/J delay allows for refocusing of chemicals shifts that differ from the carrier position Ø rotation is actually dependent on d+J o Ø 180 reverses direction and refocus rotation due to d • 1 J 13 CH ~ 125 170 Hz use average J ~ 145 Hz 13 CH of alkynes J ~250 Ø problems with Hz behaves like 13 CH 2 Ø • Decoupler is turned on during second d 2 and acquisition to collapse spins to singlet and gain NOE sensitivity • If d 2 set to 1/2 J, only observe 13 C Ø difficult average J incomplete cancellation and weak 13 C signal 13 CH 2 13 CH 3 180 o decouple

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

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

One-Dimensional (1 D) 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 t simultaneous polarization transfer for all carbons present in molecule • Common module of multidimensional NMR experiments t 90 o 180 o 90 o d 1 = recycle delay for relaxation d 2 = 1/4 J 1 H-13 C

One-Dimensional (1 D) NMR Experiments INEPT Separation in peaks indicate triplet (J~145 Hz) J -1: 1 doublet 13 CH INEPT Pascal Triangle 2 J -1: 0: 1 triplet 13 CH 2 -1: 1: 1 quartet 13 CH 3

One-Dimensional (1 D) NMR Experiments INEPT Decouple INEPT Experiment • results in selective inversion of one spin in the doublet • same result as selective polarization transfer o t during first d 2 = 1/4 J each spin moves 45 180 o 1 H refocusing pulse flips spins (would refocus after another 1/4 J delay o 1 t 180 X pulse exchanges a and b H spins – X attached to a are now attached to b and vice versa – direction of rotation is reversed o o t During second d 2, each spin moves another 45 and are aligned 180 to each other 0 t 90 X pulse generates X FID with polarization transfer t phase cycling of receiver can alternatively add and subtract spectra t Final 1 H 90 o will place one spin as +z and the other as –z Effectively, a selective 180 o on one spin

One-Dimensional (1 D) NMR Experiments INEPT Effect of INEPT Pulse Sequence on 1 H spins • because spins are 180 o to each other, turning on decoupler will cancel spins no signal • insert 180 o refocusing pulse separated by d 3=1/4 J delay 180 o refocusing pulse X spin state after standard INEPT (p 6) Decoupler turned on X collapse to singlet

One-Dimensional (1 D) NMR Experiments INEPT Refocused INEPT can Distinguish CH, CH 2 and CH 3 • selection of d 3 as a function of 1/J determines what spins are observed only 13 C attached to 1 H are observed Ø 0. 125/J optimal for all positive signal 13 CH observed Ø 0. 25/J only Ø 0. 375/J CH 2 are anti phase (negative) • common component of multidimensional NMR pulse sequences to select desired correlations • INEPT not commonly used to select spin systems DEPT Ø INEPT is too sensitive to JXH variations Ø CH

One-Dimensional (1 D) NMR Experiments DEPT Pulse Sequence of Choice to Edit 13 C NMR Spectra • not possible to use a simple vector model to explain pulse sequence involves creating multiple quantum coherence • variable p 3 pulse selects desired spin system and phase o Ø 45 pulse: CH, CH 2 and CH 3 are all positive o Ø 90 pulse: only CH signal observed o Ø 135 pulse CH and CH 3 positive with CH 2 being negative • Addition and subtraction of DEPT 45, DEPT 90 and DEPT 135 can generate spectra that only contains CH, CH 2 or CH 3 signals Ø 90 o d 1 = recycle delay for relaxation d 2 = 1/2 J 1 H-13 C 180 o ao

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

One-Dimensional (1 D) NMR Experiments W 1 A bb W 2 ab W 1 X W 0 W 1 X DEPT ba W 1 A aa Wo, W 2: multiquantum, forbidden transitions multiple quantum vector does not change during t 13 C 90 o creates multiple quantum coherence 180 o pulse refocus chemical shifts Anti-phase component (amplitude function of sin q) Last 1 H pulse Multiquantum component (amplitude function of cos q)

One-Dimensional (1 D) NMR Experiments PENDANT Pulse Sequence of Choice to Edit 13 C NMR Spectra • DEPT does not observe non protonated 13 C atoms • PENDANT same sensitivity as DEPT 13 C, 13 CH and 13 CH Ø observes quaternary 2 3 13 Ø quaternary C signals are stronger than in JMOD Ø C/CH 2 are opposite phase of CH/CH 3 signals • PENDANT with chemical shift information generally sufficient to assign 13 C spectrum Ø ambiguities can be removed with the appropriate DEPT experiment • Only requires collecting one spectrum 1 13 C spectrum Ø pointless to acquire simple H decoupled Ø replaces JMOD and APT • Again, simple spin vector diagrams are insufficient to describe pulse sequence Ø Creating multiple quantum coherence 90 o d 1 = recycle delay for relaxation d 2 = 1/4 J 1 H-13 C d 3 = 5/8 J 1 H-13 C 180 o

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

One-Dimensional (1 D) NMR Experiments INADEQUATE Detects Carbon-Carbon Coupling • 13 C nuclei only 1. 08% abundant weak satellites on either side of strong center peak 13 C is 1. 17 e 2% Ø probability of two bonded atoms both being • Experiment suppresses strong center peak to detect 13 C satellites Ø Center peak off-scale 13 C satellites Identifying 13 C-13 C connectivity beneficial for NMR assignment of complex molecules.

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

One-Dimensional (1 D) NMR Experiments INADEQUATE d 2 = 0. 08 sec J 13 C-13 C = 3 Hz d 2 = 0. 0062 sec J 13 C-13 C = 40 Hz 13 C spectrum

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