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NMR SpectroscopyApplicationsDrug designMRIFood qualityMetabonomicsStructural biology

NMR SpectroscopyBasic PrinciplesN.M.R. Nuclear Magnetic ResonanceSpectroscopic technique, thus relies on the interaction between material and electromagnetic radiationThe nuclei of all atoms possess a nuclear quantum number, I. (I 0, always multiples of .)Only nuclei with spin number (I) 0 can absorb/emit electromagnetic radiation.Even atomic mass & number: I 0 (12C, 16O)Even atomic mass & odd number: I whole integer (14N, 2H, 10B)Odd atomic mass: I half integer (1H, 13C, 15N, 31P) The spinning nuclei possess angular momentum, P, and charge, and so an associated magnetic moment, . x PWhere is the gyromagnetic ratio

NMR SpectroscopyBasic PrinciplesB0The spin states of the nucleus are quantified:I, (I - 1), (I - 2), , -I I (e.g. 1H)Energy– – E h h B0/2 B0 0B0 0

NMR SpectroscopyBasic PrinciplesIn the ground state all nuclear spins are disordered, andthere is no energy difference between them. They aredegenerate.BoSince they have a magnetic moment, when we apply astrong external magnetic field (Bo), they orient eitheragainst or with it:There is always a small excess of nuclei (populationexcess) aligned with the field than pointing against it.

NMR SpectroscopyBasic Principles Bo 0 Bo 0 E h 0 h B0/2 0 is the Larmor Frequency 0 B0, angular velocityzB0 yx

NMR SpectroscopyBasic PrinciplesEach level has a different population (N), and the difference between the two is relatedto the energy difference by the Boltzmman distribution:N /N e E/kT E for 1H at 400 MHz (B0 9.5 T) is 3.8 x 10-5 Kcal/mol N /N 1.000064The surplus population is small (especially when compared to UV or IR).That renders NMR a rather insensitive technique!

NMR SpectroscopyThe electromagnetic spectrum -rays60010221020Mossbauer500400X-raysvisible 1014infraredelectronic100rotational108NMR106 equency8201012microwave19F3001018ultraviolet 1016101H13C /MHzolefinicacetylenicaliphatic /ppm

NMR SpectroscopyThe Vector Model M0yxyxyyxx

NMR SpectroscopyNMR excitationzB1 C * cos ( ot)MoyB1xBoiTransmitter coil (x)B1 is an oscillating magnetic fieldyy- 0 0 xx

NMR SpectroscopyLaboratory vs. Rotating framezz’ 0 M0M0y 0 xy’B1- 0 Laboratory framex’-2 0 Rotating frame

NMR SpectroscopyEffect on an rf pulsezzM0 1tp (degrees) 90 B1xyyB1x 180

NMR SpectroscopyMagnetization properties 1H 400,000,000 Hz A 400,000,005 HzzAyyB1MAyMAx 5 Hzxxyxxyxyxy

NMR SpectroscopyMagnetization properties 1H 400,000,000 Hz A 400,000,005 HzzAB1MAyyyyMAx 5 HzxxxMAy MA cos tdetectorMAx MA sin tIMIMMAMAttt

NMR SpectroscopyThe Fourier TransformFTtime domainIMfrequency domain 1/ t tFTttimefrequency

NMR SpectroscopyThe Fourier TransformIMSignal Induction Decay (FID)time

NMR SpectroscopyThe Fourier TransformFT

NMR SpectroscopyContinuous wave vs. pulsed NMR o or Bo o or Botime

NMR SpectroscopyContinuous wave vs. pulsed NMR For cos( t)FT absorptive lines For sin( t)FT despersive lines

NMR SpectroscopyContinuous wave vs. pulsed NMRA monochromatic radiofrequency pulse is a combination of a wave(cosine) of frequency 0 and a step function* tp Since f 1/t, a pulse of 10 sduration excites a frequencybandwidth of 105 Hz!FT o

NMR SpectroscopyContinuous wave vs. pulsed NMR E t h or t 1

NMR SpectroscopySingle-channel signal detectionz yxFT 0 -

NMR SpectroscopyQuadrature detectionz yxMyMxMyMx

NMR SpectroscopyQuadrature detectioncosysinx 0 - 0 HzHz

NMR SpectroscopyThe Chemical ShiftThe NMR frequency of a nucleus in a molecule is mainly determined by itsgyromagnetic ratio and the strength of the magnetic field BThe exact value of depends, however, on the position of the nucleus in the molecule ormore precisely on the local electron distributionthis effect is called the chemical shift

NMR SpectroscopyThe Chemical Shift E h h B/2 Nuclei, however, in molecules are never isolated from other particles that are chargedand are in motion (electrons!). Thus, the field actually felt by a nucleus is slightly different from that of the appliedexternal magnetic field!!

NMR SpectroscopyThe Chemical Shift E h h Be /2 Beff, is given by B0-B B0-B0 B0(1- ) and is the chemical shift ( - ref) ref B0(1- )2 106 106 ( ref- )

NMR SpectroscopyThe Chemical Shiftmethylprotonsamide protons protonsmethyleneprotonsaromatic ring750 MHz 1H spectrum of a small protein shieldingfrequencymagnetic field

NMR SpectroscopyThe Chemical Shift

NMR SpectroscopyThe Chemical Shift

NMR SpectroscopyNuclear Shielding dia para nb rc ef solvdiamagnetic contributionparamagnetic contributionneighbor anisotropy effectring-current effectelectric field effectsolvent effect

NMR SpectroscopyNuclear Shielding - diamagnetic contributionThe external field B0 causes the electrons to circulate within their orbitalsB0h B0h B0(1- )B’The higher is the electron density close to the nucleus, the larger the protection is!

NMR SpectroscopyNuclear Shielding - diamagnetic contributionDepends on the electronegativityCH3X

NMR SpectroscopyNuclear Shielding - paramagnetic contributionThe external field B0 mixes the wavefunction of the ground state with that of the excited stateThe induced current generates a magnetic field that enhances the external field and deshields thenucleusLUMOB0HOMO p 1 1R3

NMR SpectroscopyChemical shift range1H; 10 ppm13C; 200 ppm19F; 300 ppm31P; 500 ppmLocal diamagnetic and paramagnetic currents make only modest contributions to 1H shielding!

NMR SpectroscopyChemical Shift AnisotropyNuclear shielding, , is a tensor.The distribution of the electrons about the nucleus is non-sperical- thus, the magnitude of theshielding depends on the relative orientation of the nucleus with respect to the static field.In isotropic cases: ( 11 22 33)In static cases, e.g. solid state

NMR SpectroscopyNuclear Shielding - neighboring groupB0BABμpar μperAμpar μper --

NMR SpectroscopyNuclear Shielding - neighboring group -CC-μpar μperC μpar μper C-C 2H 476C 2H 2543C 2H 6210ppm

NMR SpectroscopyNuclear Shielding - ring-current effectMore pronounced in aromatic rings due to the electron cloudsBo9.28e--2.99

NMR SpectroscopyNuclear Shielding - hydrogen bondingHydrogen bonding causes deshielding due to electron density decrease at the proton site[EtOH] in CCl4CH3CH2OH1M0.1M0.01M0.001M6420ppm

NMR SpectroscopySpin-spin (scalar) couplingHF (1H-19F)HFJHFJHF

NMR SpectroscopySpin-spin (scalar) couplingHF (1H-19F)H FHH FBo19F1HH FHH F19F1HNuclear momentMagnetic polarizationof the electronE h JAX mA mXwhere m is the magnetic quantum numberJAX is the spin-spin coupling constant

NMR SpectroscopySpin-spin (scalar) couplingAX2AMXAX3

NMR SpectroscopySpin-spin (scalar) couplingStrong coupling – 10 J

NMR SpectroscopySpin-spin (scalar) couplingThe principal source of scalar coupling is an indirect interaction mediated by electrons involved in chemical bondingThe magnitude of interaction is proportional to the probability of finding the electron at the nucleus (R 0)Magnitude in Hz- independent of the external magnetic fieldH3C – CH3H2C – CH2HC CH125 Hz160 Hz250 Hz

NMR SpectroscopySpin-spin (scalar) couplingThree-bond coupling most useful since it carries information on dihedral anglesEmpirical relationship: the Karplus relation3J A B cos C cos2

NMR SpectroscopyChemical shifts on the rotating frame500 MHzz3y2 0x tzyx 500 Hz

NMR SpectroscopySpin couplings on the rotating frameJzy 0x tz -J/2 Hzyx J/2 Hz

NMR SpectroscopyThe basic spin-echo pulse sequence90 appliedalong x axis180 appliedalong y axisx delayy acquisition

NMR Spectroscopy90 appliedalong x axisEffect of spin echo on chemical shift evolution180 appliedalong y axisx y acquisitiondelayzzy90xy xxzzyzyx180y Axy x A

NMR Spectroscopy90 appliedalong x axisEffect of spin echo on scalar coupling evolution180 appliedalong y axisx y acquisitiondelayzzy90x1H-Xy xxzz-J/2yz(only180yyy -J/2x J/2x J/2x1H)

NMR Spectroscopy90 appliedalong x axisEffect of spin echo on scalar coupling evolution180 appliedalong y axisx y acquisitiondelayzzy90x1H-Xy xxzz-J/2z180yyy J/2x J/2x-J/2xy(both 1H and X)

NMR SpectroscopyWater suppression by the Jump and Return method zzy90xy xxzzyzy A90-xxyxx

NMR SpectroscopyWater suppression

NMR SpectroscopySpin decouplingdecouple1H13CH CHH CH CHH C

NMR SpectroscopyThe J-modulated spin echodecouple1Hyx13C

NMR SpectroscopyThe J-modulated spin echodecouple1Hyx13C

NMR SpectroscopyThe J-modulated spin echoIf 180J degreesC:CH:CH2:CH3:I 1I cos I cos2 I cos3 decouple1Hyx13C

NMR SpectroscopyThe J-modulated spin echodecouple1Hyx13C 1/J13C(ppm)

NMR SpectroscopySensitivity enhancementNMR has poor sensitivity compared to other analytical techniquesThe intrinsic sensitivity depends upon the gyromagnetic ratio, A greater contributes to:a high resonant frequency- large transition energy difference- greater Boltzmann population differencehigh magnetic moment and hence a stronger signalhigh rate of precession which induces a greater signal in the detection coilSo, the strength of NMR signal is proportional to 3Noise increases a square-root of observed frequency}S/N 5/2

NMR SpectroscopySensitivity enhancement by polarization transferSignal sensitivity enhancement by transferring the greater population differences of high spins onto their spin-coupled low- partners. C21H-13CH2H1 C1 spin pair

NMR SpectroscopySensitivity enhancement by polarization transferSignal sensitivity enhancement by transferring the greater population differences of high spins onto their spin-coupled low- partners. C2 C C2 H2 C2 2 H1C2H2 C 2 CC1 C2 2 C2 H1 C1(inverted)2 C C 2 2 C C CC1H1H1H2C1C2H2C2-3:5

NMR SpectroscopySensitivity enhancement by polarization transferSignal sensitivity enhancement by transferring the greater population differences of high spins onto their spin-coupled low- sed,decoupledINEPT

NMR SpectroscopyRelaxationWhen perturbed, the nuclear spins need to relax to return to their equilibrium distributionE.g. when the sample is put into a magnet, the Boltzmann distribution of spins among the energylevels changes due to a change in the energy of the various levelsE.g. after applying electromagnetic radiation, which induces transitions between energy levels,the system returns to its equilibriumThis process is called relaxation

NMR SpectroscopyLongitudinal Relaxation: Establishing Equilibriumzyxzzyyxxzzxx

NMR SpectroscopyLongitudinal Relaxation: Establishing EquilibriumRecovery of the z-magnetization follows exponential behaviordMzdt (M0-Mz)T1Mz M0 (1-2e-t/T1)where T1 is the longitudinal relaxation time

NMR SpectroscopyLongitudinal Relaxation: Measurementxxzzz180xz yx 90xyxyyxxzz 90xyxyx

NMR SpectroscopyLongitudinal Relaxation: Measurementxx

NMR SpectroscopyLongitudinal Relaxation: Exponential growthMz M0 (1-2e-t/T1)By the end of 5T1 sec, the magnetization has recovered by 99.33%

NMR SpectroscopyLongitudinal Relaxation: optimizing sensitivity

NMR SpectroscopyLongitudinal Relaxation: optimizing sensitivity

NMR SpectroscopyLongitudinal Relaxation: optimizing sensitivityoptimum pulse repetition time when using 90ºQuantitative measurements and integration

NMR SpectroscopyTransverse Relaxation: magnetization loss in the x-y planeyx---yyy xxtimex

NMR SpectroscopyTransverse Relaxation: magnetization loss in the x-y plane 1 T2*

NMR SpectroscopyTransverse Relaxation: Measurementxz z z90x- yyyx xz180y xyzy yxx

NMR SpectroscopyTransverse Relaxation: Measurement

NMR SpectroscopyT1 vs T2 RelaxationT1 T2For small molecules, T1 T2For large molecules, T1 T2Longitudinal relaxation causes loss of energy from the spins (enthalpic)Transverse relaxation occurs by mutual swapping of energy between spins (entropic)

NMR SpectroscopyRelaxation mechanismsNuclear spin relaxation is not a spontaneous process; it requires stimulation bysuitable fluctuating fields to induce the necessary spin transitionsTwo main mechanismsDipole-dipoleChemical shift anisotropy