Magnetisation Transfer Schemesiupab/madhu2.pdfMagnetisation Transfer Schemes P. K. Madhu Department...

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Magnetisation Transfer Schemes P. K. Madhu Department of Chemical Sciences Tata Institute of Fundamental Research Homi Bhabha Road Colaba Mumbai 400 005, India

Transcript of Magnetisation Transfer Schemesiupab/madhu2.pdfMagnetisation Transfer Schemes P. K. Madhu Department...

  • Magnetisation Transfer Schemes

    P. K. MadhuDepartment of Chemical Sciences

    Tata Institute of Fundamental ResearchHomi Bhabha Road

    ColabaMumbai 400 005, India

  • Sensitivity of NMR Spectroscopy

    S/N∼ NγexcγdetB3/20 NST1/22

    S/N signal-to-noise ratio

    N number of spins

    gyromagnetic ratio of excited spins

    gyromagnetic ratio of detected spins

    static magnetic field

    NS number of scans

    transverse relaxation time

    γexc

    γdet

    B3/20

    T1/22

    sample concentration

    isotope labeling

    magnet size

    measurement time

    molecular weight

  • Spin-1/2 nucleus

    |α>

    |β>

    ΔE=(h/2π) γBFor 1H:γ=26.75 rad T-1s-1ΔE=2.65*10-25J for B=9.4 TkT= 4.14*10-21 J

    ΔE/kT=6.4*10-5

    Sensitivity of NMR Spectroscopy

    Preferred Nβ

  • Spin-1/2 nucleus

    |α>

    |β>

    ΔE=(h/2π) γBFor 1H:γ=26.75 rad T-1s-1ΔE=2.65*10-25J for B=9.4 TkT= 4.14*10-21 J

    ΔE/kT=6.4*10-5

    Sensitivity of NMR Spectroscopy

    Preferred Nβ

  • 2 million

    1 million+16

    1 million+64

    1 million

    1 million

    1 million

    1 million+128

    ΔE

    B0 (Tesla)

    0 T 2.35 T 9.4 T 18.8 T

    Energy Levels, Magnetic Field, and Relative Population

    Spin-1/2 nucleus

  • NMR Active Nuclei: Properties

  • NMR Concentrations

    Can the polarisation from an abundant spin, like 1H, betransferred to a rare spin, like 13C?

    Polarisation Transfer

    Sensitivity of NMR Spectroscopy: How to Increase?

    Higher magnetic fields

    Lower temperatures Cryoprobes/sample cooling

    Hyperpolarised NMR Transfer of abundant population from some source to rare nuclei

  • Selective Population Transfer (SPT)

    Consider two proton spins, homonuclear 1H-1H spin system, weakly J coupled(having a large chemical-shift difference), forming an AX spin system

    αα

    αβ βα

    ββ

    A Xx

    X

    A

    A

    2 3

    1

    4 1,2 3,41,3 2,4

    • • • •

    • •• •

    RF irradiation leading to saturationOf 1-3 transition

    αα

    αβ βα

    ββ

    A X

    X

    X

    A

    A

    2 3

    1

    4

    1,2

    3,4

    1,3 2,4

    • • •

    • • •• • 3-4 transition gets

    a 50% increase

    Population is transferred from one nuclues to the other

  • Selective Population Inversion (SPI)

    180 90

    Soft pulse, transition selective

    αα

    αβ βα

    ββ

    A XX

    X

    A

    A

    2 3

    1

    4 1,2 3,41,3 2,4

    • • • •

    • •• •

    Soft pulse, transition selective

    αα

    αβ βα

    ββ

    S

    S

    I

    I

    2 3

    1

    4

    1,2

    3,4

    1,3

    2,4

    • •

    • • • •• •

    3-4 transition getsa two-fold increase

  • Polarisation Transfer

    Both SPT and SPI can lead to polarisation transfer, but we are onlydealing with homonuclear spin systems, not really interesting

    SPT and SPI can identify scalar coupled spin systems in crowdeddpectral regions

    But the real use of these are in heteronuclear spin systems

  • SPI in Heteronuclear Spin Systems1,2 3,4

    180, soft pulseon 1H

    Overall 13C intensity:Before perturbation=2+2=4And after pertrubation=6+10=16Four-fold enhancement!

    13C2,4

    αα

    αβββ

    13C

    1H

    1H

    βα• • • •• • • •

    1

    4

    3

    2

    1,2

    3,4

    1,3

    A X

    • • • • •• • • • •

    • • 13C1H

    αα

    αβββ13C

    13C

    1H

    1H

    βα• • • •• • • •

    • • • • •• • • • •

    • •

    1

    4

    3

    2

    1,3 2,4

    A X

    13C1H

  • SPI in Heteronuclear Spin Systems

    By manipulating the polarisation of the protons, we have accomplished a four-fold enhancement for 13C signals, counting both positive andnegative signals

    The factor of 4 comes from γH/γC ratio; it will be 10 for 1H to 15N polarisation transfer

    This is all fine, but we have up and down signals, not quite interesting

    2,4

    1,2

    3,4

    1,3

    A X

    13C1H

    Overall 13C intensity:Before perturbation=2+2=4And after pertrubation=6+10=16Four-fold enhancement!

  • Echo Modulations

    x

    y

    α

    β

    AX spin system, heteronuclear

    18090

    A

    X

    MAXα

    MAXβx

    y

    MAXα

    MAXβ

    x

    y

    MAXα

    MAXβ

    x

    y

    α

    β

    MAXα

    MAXβ

    τ τ

    Everything is refocussed, chemical shifts, RF and B0 inhomogeneities, andcoupling (scalar) effects- The spin-echo phenomenon

  • Echo Modulations

    x

    y

    α

    β

    AX spin system, homonuclear

    18090

    A

    MAXα

    MAXβx

    y

    MAXα

    MAXβ

    x

    y

    MAXβ

    MAXα

    x

    y

    MAXβ

    MAXβ

    τ τ

    No J refocussing

    φ

    (2 ) 4 AXJφ τ π τ=The difference in angular frequency between the two components is 2πJAX

    180

    X τ τAX spin system, heteronuclear

  • x

    y

    x

    y

    tD = 1 / 2JJ / 2

    NO REFOCUSSING REFOCUSSINGBEFORE DECOUPLING BEFORE DECOUPLING

    J-Modulation and Polarisation Transfer

    13C magnetisation vectors,+5 and -3 in length in thexy plane

    180

    90

    tp

  • J-Modulation and Polarisation Transfer

    180

    90x

    τ τ

    A, 1H

    τ=1/4J

    X, 13C

    13C signal of lengths -3 and 5 created along the z-axis

    x

    yMXAα

    MXAβ

    τ=J/4x

    y

    900x

    180

    180x

    180xA

    180xXx

    τ=J/4x

  • J-Modulation and Polarisation Transfer

    We achieve polarisation transfer and signal enhancement, but:

    •The proton 180 pulse has to be selective•Lack of generality•The need is to set up appropriate polarisation of all the protontransitions regardless of frequency/selectivity

    Hence, we need a pulse sequence that generates anti-phaseproton transition for every 1H-13C spin pairs, but non-selectively

  • INEPT

    Insenstive nuclei enhanced by polarisation transfer

    90x

    τ τA, 1H

    X, 13C

    180x 90y

    180x 90x

    τ=1/4J

    The idea is to create an antiphase doublet for the proton magnetisationand then a 90 pulse on 13C will create the (-3,5) carbon magnetisation

  • x

    y

    z

    90x

    τA, 1H180x

    τ

    X, 13C

    90y

    180x 90x

    90xA

    Monitor the 1H magnetisation vectors

    τ=J/4

    x

    yz

    900

    x

    y

    z180xA,X

    τ=J/4

    x

    y

    z

    90yA

    x

    y

    z

    Anti-phase proton magnetisation and the subsequent90 on 13C creates the (-3,5) carbon vectors as earlierHere, we achieve uniform polarisation transfer

    INEPT

  • 180xA

    180x

    X

    δ δ

    a b

    90y

    90x

    c

    12 ( )4

    INEPTx z y

    AX

    A A XJ

    δ⎯⎯⎯→ =

    INEPT

    2,4

    1,3

    13C

    Factor of 4 as enhancement

  • INEPT

    Az

    -Ay

    -Ay cosπJδ

    Ay cosπJδ

    Ay cosπJδ

    2AxXz sinπJδ

    2AxXz sinπJδ

    -2AxXz sinπJδ

    Ay cos2 πJδ -2AxXz cosπJδ sinπJδ -2AxXz sinπJδ cosπJδ -Ay sin

    2 πJδ

    Ay cos2 πJδ -Ay sin

    2 πJδ-4AzXz cosπJδ sinπJδ

    Ay cos2 πJδ -4AzXy cosπJδ sinπJδ

    -Ay sin2 πJδ

    δ = 14J

    0.5Ay -0.5Ay2AzSy

    90x

    δA, 1H

    X, 13C

    180x

    δ

    90y

    180x 90x2πJAzXz

    90Ax

    180Ax

    180Xx

    2πJAzXz

    90Ay

    90Xx

    δ = 14J

  • INEPT

    • Enhances polarisation– Basic building block in most pulse schemes

    • Spectral editing– To select functional groups of our choice

    • Establishes correlation between sets of coupled spins– Most important in multi-dimensional experiments

  • INEPT

    13C coupled

    INEPT SpectrumINEPT

  • -1:1-1: 0:1 -1: -1: 1:1

    INEPT Spectral Patterns

    CH3CH2CH

    13C spectrum

  • 180xI

    180x

    S

    δ δ

    a b

    90x

    90x

    c

    Refocused NEPT

    . 1( )4

    ref INEPTx x

    IS

    I SJ

    δ⎯⎯⎯⎯→ =

    180xΔ/2 Δ/2

    d

    180x

    Refocused INEPT

    90x

    For CH spin systems, the optimum value for Δ=1/2JCH

    In case of CH, CH2, and CH3 groups, optimum value for Δ=1/3JCH

  • Behaviour of CH, CH2 and CH3 Groups

    Spectral Editing

  • 180x

    90x

    τ τθy

    τ

    180x

    90x

    I, 1H

    S, 13C

    Distortionless Enhancement by Polarisation Transfer

    DEPT

    The relative intensities of the mulitplet components in INEPT spectradiffer from the normal spectra, hence, DEPT

    In DEPT, the θ pulse takes the role of Δ in INEPT, so the t delayis set to 1/2J and depending on the values of θ one gets variousfunctional group spectra

  • DEPT45 experiment yields a positive peak for every carbon with attached protons: Ca at 16 ppm, Cb at 29 ppm, and Cd, Ce, and Cf at 128.5, 128.9, and 129 ppm, respectively. Note in the spectrum below that carbon in the CDCl3 solvent does not give a signal, since it has no attached protons

    DEPT45

  • Dept 90 yields only CH yields peaks; CH0, CH2, and CH3 are invisible. In our example we see only three lines due to Cd, Ce, and Cf in the aromatic range from 126 to 129 ppm.

    DEPT90

  • With DEPT135 CH2 yields negative peaks, whereas CH and CH3 are positive. Thus, we see Ca, Cd, Ce, and Cf as positive peaks, while Cb is negative.

    DEPT135

  • To distinguish the various multiplicity patterns in 13C NMR, three DEPT spectra are acquired

    DEPT: Spectral Editing

  • DEPT: Spectral Editing

    CH3=FID(45)+FID(135)-0.707 FID(90)

  • DEPT: Spectral Editing

  • Major Relaxation Pathways

    1. Dipole-dipole coupling

    2. Scalar coupling

    3. Chemical shift anisotropy

    4. Chemical exchange

    5. Paramagnetic interactions

    6. Spin rotation

  • NOE is the change in the intensity of an NMR resonance whenthe transitions of a dipolar coupled spin are perturbed (saturated/inverted)

    The NOE enhancement of I spin upon saturating S spin is defined as

    0

    0

    { }II IS

    Iη −=

    Equilibrium I intensity

    Perturbed spin

    Observed spin

    Nuclear Overhauser Effect

  • αα (∗∗∗)

    ββ (∗)

    (∗∗∗) αβ

    W1X

    W1X

    W1A

    W1A

    βα (∗)W2AX

    NOE: Transition Probabilities

    W0AX

    W0AX and W2AX are determined by dipolar couplings andhave a distance dependence, r-6, and rotational correlationtime dependence, τc

  • NOE and Molecular Motion

    Relaxation

    W2 W1 W0

    Depends on the strength of the local (dipolar) fields fluctuating at that frequency, ω

    Depends on the molecular motion at that frequency, ω

    W0 transition will be predominant when the molecules tumble at ωA-ωX frequency, kHz, for large moleculesW1 for molecules tumbling at Larmor frequenciesW2 for molecules tumbling at twice the Larmor frequencies, small molecules, fast tumbling

    Small molecules lead to positive NOEBig molecules lead to negative NOESomewhere in between null NOE

  • W2AX - W0AX

    2W1X + W2AX + W0AX ηA = = fA{X}

    σAX = W2AX - W0AXρAX = 2W1X + W2AX + W0AX

    ηA = σAX / ρAX= fA{X}

    σAX = W2AX - W0AXρAX = 2W1X + W2AX + W0AX

    ηA = σAX / ρAX= fA{X}

    NOE: Some Expressions

    Wn ∝ 1r6 J(nω)

    J(nω) = τc1+(nωτc)2

  • Steady-State NOE

    ωτc1

    ωτc=1.12

  • Hb

    Ha

    Hc

    HbHa Hc

    _ =ηab ηac

    C

    NOE Difference Spectroscopy

    13C

    1H

    Steady-state NOE

    Knowing a reference distance, other distances may be calculated

    ηab ∝ rab-6rac = rab * ( ηab / ηac ) -1/6

    ηac ∝ rac-6

    ηab ∝ rab-6rac = rab * ( ηab / ηac ) -1/6

    ηac ∝ rac-6

  • αα (∗∗)

    ββ (∗∗)

    (∗∗∗∗) αβ

    W1X

    W1X

    W1A

    W1A

    βα ()W2AX

    W0AX

    180X90

    selective inversion

    Transient NOE

    τm

    τm

    Inte

    nsity

    Monitor the magnetisation of the dipolarcoupled spin by inverting the other spinas a function of the mixing time. The initialrate of growth is proportional to r-6

    Steady-state NOE could give ambiguous results in big molecules due to othermagnetisation transfer processes, such as, spin diffusion. Hence, transient NOEmuch more desirable and useful

  • Nuclear Overhauser Effect

  • Nuclear Overhauser Effect

  • Nuclear Overhauser Effect

  • Nuclear Overhauser Effect

  • Nuclear Overhauser Effect

  • • Useful to identify spins undergoing cross-relaxation

    • Direct dipolar couplings provide primary means of cross relaxation

    • Cross relaxation manifests in the form of cross peaks in the NOESY spectrum

    Nuclear Overhauser Effect

  • Overhauser being awarded the National Medal of Science, 1994

  • "Overhauser proposed ideas of startling originality, so unusual that they initially took portions of the scientific community back, but of such depth and significance that they opened vast new areas of science."

    The consequences of this discovery---known as the Overhauser Effect---for nuclear magnetic resonance, and through nuclear magnetic resonance for chemistry, biology and high-energy physics have been enormous. The idea, which has also had very practical consequences, was so unexpected that it was originally resisted vehemently by the authorities in the field. Not until its existence was demonstrated experimentally by Slichter and Carver in 1953 was it fully accepted. It has been said that one can judge the importance of a new discovery in physics by the number of other fields of science and engineering it impacts. From this point of view this contribution of Overhauser ranks among the highest.

  • When first proposed as a contributed paper at an APS meeting in April 1953, the proposal was met with much skepticism by a formidable array of physics talent. Included among these were notables such as: Felix Bloch (recipient of 1952 Physics Nobel Prize), Edward M. Purcell (recipient of Nobel Prize 1952 with Bloch and session chair), Isidor I. Rabi (recipient of Physics Nobel Prize, 1944) and Norman F. Ramsey (recipient of Physics Nobel Prize, 1989). Eventually everyone was won over. In a letter dated 27 July 1953, Norman F. Ramsey stated the matter succinctly2,3:

  • July 27, 1953Dear Dr. Overhauser:

    You may recall that at the Washington Meeting of the Physical Society, when you presented your paper on nuclear alignment, Bloch, Rabi, Pearsall, and myself all said that we found it difficult to believe your conclusions and suspected that some fundamental fallacy would turn up in your argument. Subsequent to my coming to Brookhaven from Harvard for the summer, I have had occasion to see the manuscript of your paper.

    After considerable effort in trying to find the fallacy in your argument, I finally concluded that there was no fundamental fallacy to be found. Indeed, my feeling is that this provides a most intriguing and interesting technique for aligning nuclei. After considerable argument, I also succeeded in convincing Rabi and Bob Pound of the validity of your proposal and I have recently been told by Pound that he subsequently converted Pearsall shortly before Pound left for Europe.

    I hope that you will have complete success in overcoming the rather formidable experimental problems that still remain. I shall be very interested to hear of what success you have with the method.

    Sincerely,Norman F. Ramsey

  • April 20, 1993Dear Al:

    I greatly appreciate your thoughtful remarks about the letter I wrote you forty years ago. Although I clearly remember surprising some of my friends by writing a very favorable referee report, I had forgotten that I also had written you a letter. You might be interested in how I came to get the matter straight and avoid the lifelong embarrassment of being responsible for the rejection of a great pioneering paper.

    After the APS meeting I did not understand your paper and was thoroughly convinced by the vigorous arguments of Bloch, Rabi and others that a radio frequency field always produces heating. I was consequently annoyed when I was asked to referee the paper and therefore would have to find exactly what was wrong. I started my study with strong prejudices against you but I then remembered that in high school physics I had always had trouble remembering how a Servel (gas) refrigerator worked. I decided that I could not write a negative referee report until I understood once again how the Servel worked. By the time I understood that, I had lost my prejudice against your paper and on further study was convinced you were right. Incidentally the easiest way for me to remember how in principle a gas refrigerator can work without violating thermodynamics is to remember one could use the heat of the gas flame to operate a steam engine which in turn could operate a mechanical refrigerator.

    Sincerely yours,Norman F. Ramsey

  • NORMAL, NO NOE

    INEPT

    REFOCUSSED INEPT

    REFOCUSSED INEPT AND

    DECOUPLING

    NORMAL DECOUPLING

    FULL NOE

    13C Chloroform Spectra

  • INEPT

    29Si with INEPT Scheme

  • Enhancement via

    NOE T1 of interest is that of observed nucleus

    INEPT T1 of interest is that of proton

    INEPT and NOE Transfers

    INEPT

    NOE

    I=I0γAγX

    I=I0(1 +γA2γX

    )

  • Signal strength available by direct observation in the presence of full NOE from protons and from polarisation transfer from protons to the heteronucleus

    INEPT and NOE Transfers

    Nucleus Maximum NOE Polarisation Transfer

    31P 2.24 2.47

    13C 2.99 3.98

    29Si -1.52 5.03

    15N -3.94 9.87

    57Fe 16.48 30.95

    103Rh -14.89 31.78

  • • Time scales and molecular motions

    Atomic fluctuations, vibrations. Influences bond length measurementsGroup motions. (covalently linked units) Molecular rotation, reorientation Relaxation, linewidths, correlation timesMolecular translation, diffusion DOSY NMRRotation of methyl groups. 2H NMRFlips of aromatic rings. 2H NMRDomain motions. 2H NMR

    Chemical exchange, proline isomerization Chemical shiftsAmide exchange 15N-1H HSQCLigand binding Transferred NOE measurements

    Dynamics and Relaxation

  • s ns ps fsμsmsFastSlow Very slow

    Slow Fast Very fast Ultra fast

    MacroscopicDiffusion,Flow

    Chemical exchange

    Molecular rotations

    Molecular vibrations

    Motional Timescales

  • Chemical Exchange

    Motional process leading to formation or rupture of chemical bonds: Chemical exchange

    The electronic structure is different in both the forms leading to differencechemical shifts and coupling constants when the exchange processtakes place: Detectable by NMR provided the process is on an appropriatetime scale

  • Conformationalequilibrium

    Chemicalequilibrium

    Kex

    KB

    NMR and Dynamic Processes

    This could be a chemical reaction, conformational equilibrium, exchange between the bound and free states of a ligand/protein complex, ligand binding of drugs to proteins.

  • N H

    O

    N H

    O

    NMR: Measurement of Rate Constants

    Inversion of NN-dimethylformamide

    1 1Rate (s) >> or

    δr - δb Δδ

    1 1Rate (s) >> or

    δr - δb Δδ

    The two methyl groups exchange due to the double-bond nature of the amide bond.They give two distinct resonance lines as long as the rate of exchange is longerthan the relative difference in frequency of the two resonances

  • Lets now start increasing the temperature. Since the rate depends on the ΔG of theinversion, and the ΔG is affected by T, higher temperature will make things go faster. What we see in the NMR looks like this:

    At a certain temperature, called the coalecense temperature,the rate of the exchange between the two species becomescomparable to the difference in chemical shifts of the sites:

    Past this point, the NMR measurement cannot distinguishbetween things in either site, because things are exchangingfaster than the difference in relative frequencies.

    T TC

    1 1Rate (s) ≤ or

    δr - δb Δδ

    1 1Rate (s) ≤ or

    δr - δb Δδ

    NMR: Measurement of Rate Constants

  • Δδ * Rate > 1 Slow exchange

    Δδ * Rate = 1 Transition

    Δδ * Rate < 1 Fast exchange

    NMR: Measurement of Rate Constants

    Now, since we can estimate the temperature at which we have the transition taking place, we can get thermodynamic and kinetic data for the exchange process taking place.

    If we did a very detailed study, we see that we have to take into account the populations of both sites (one site may be slightly favored over the other energetically), as well as the peak shape.

    Assuming equally populated sites (equal energies) simple relationshipscould be obtained.

  • NMR: Measurement of Rate Constants

    From the Δδ value (in Hz) at the limit of slow exchange we estimate the rate constant at the coalecense temperature:

    Since we have the coalecense temperature, we can calculate the ΔG‡ of the process:

    With NMR we can measure rates from 10-2 to 108 s-1.

    Kex = π * Δν / √2 = 2.22 * ΔνKex = π * Δν / √2 = 2.22 * Δν

    ΔG‡ = R * TC* [ 22.96 + ln ( TC / Δν ) ]ΔG‡ = R * TC* [ 22.96 + ln ( TC / Δν ) ]

  • +

    FreeBound

    Ligand Conformation: Transfer NOE

    Ligand binding to a receptor?

    Eg. Drug binding to protein, helpful in the design of drugs providedthe chemical requirements of activity and conformational requirements of binding are known

    Often the bound ligand-receptor form cannot be solved as theprotein could be very large.

    Monitor the NOE rates!

  • Ligand Conformation: Transfer NOE

    *

    *

    HS

    HI

    When bound, the protons in the marked carbons will have an NOE interaction. It will be very hard to see it with the protein also having tons of other NOE correlations

    HS

    HI

    *

    *

    H

    H

    *

    *

    HS

    HI *

    *

    koff kunf

    Usually, koff

  • bound L free L

    protein

    Ligand Conformation: Transfer NOE

    Besides retaining NOE, sharp NMR spectral lines of the ligand could beobtained outside of the protein

    The ligand cannot bind tightly to the receptor (we need constant exchange between bound and free ligand).

    The koff rate has to be much smaller than the spin-lattice relaxation rate, otherwise the NOE dies before we can detect it.

    Size of the receptor is not an issue.

  • •Correlation experiments, homocorrelation/heterocorrelation•Assignments, connectivities•Single-quantum/multiple-quantum correlation

    Correlation Experiments: Magnetisation Transfer

    Magnetisation transfer

    Coherent Incoherent

    Mediated via dipolar couplings, NOE/ROE/chemical exchange

    Mediated via scalar couplingsthrough one or more coherenttransfer steps

    Essentially four building blocks: COSY, TOCSY, INEPT, and HMQC

  • Building Blocks, Spin Echo Schemes

    180xI

    180xI

    180x

    S

    τ τ

    a b c dτ τ

    a b c d

    a b c d

    I

    S

    180xDec. CS

    Dec. SE

    JIS SE

    .Dec SEx xI I⎯⎯⎯→

    . 142 ( )IS IS

    J SEx y z JI I S τ⎯⎯⎯→ =

    . cos( 2 ) sin( 2 )Dec CSx x I y II I Iτ τ⎯⎯⎯→ Ω + Ω

  • Heteronuclear Multiple-Quantum Correlation, HMQC

    HMQC building block which takes as input transverse I magnetisation and frequency labels it with S

    Essentially HMQC does the following:

    cos( )HMQCx x sI I t⎯⎯⎯→ Ω

    t/2 t/2 ΔΔ

    90x 90x

    180x

    a b c d e f

    S

    I

  • INEPT

    180xI

    180x

    S

    δ δ

    a b

    90y

    90x

    c

    INEPT

    12 ( )4

    INEPTx z y

    IS

    I I SJ

    δ⎯⎯⎯→ =

  • Refocused INEPT

    180xI

    180x

    S

    δ δ

    a b

    90y

    90x

    c

    Refocused NEPT

    . 1( )4

    ref INEPTx x

    IS

    I SJ

    δ⎯⎯⎯⎯→ =

    180xδ δ

    d

    180x

  • Reverse INEPT

    180xI

    180x

    S

    δ δ

    a c

    90y

    90x

    b

    Reverse INEPT

    . 12 ( )4

    rev INEPTz y x

    IS

    I S IJ

    δ⎯⎯⎯⎯→ =

  • Reverse Refocused INEPT

    180xI

    180x

    S

    δ δ

    a b

    90y

    90x

    c

    Reverse refocused INEPT

    180xδ δ

    d

    180x

    . . 1( )4

    rev ref INEPTx x

    IS

    S IJ

    δ⎯⎯⎯⎯⎯→ =

  • Conclusions

    •It is possible to manipulate the spin populations

    •Transfer of polarisation possible from one nucleus to another

    •Polarisation transfer mediated by J or dipolar coupling

    •In the case of dipolar coupling, NOE, distance information is present

    •These form the building blocks in experiments to determinethe structure of big molecules