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Introduction to 2D NMR

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TOtal Correlation SpectroscopY (TOCSY) HOmonuclear HArtman-HAhn spectroscopy (HOHAHA) ... ROESY (Rotating Overhauser Effect SpectroscopY) dH. dH. MW = 800 Da ... – PowerPoint PPT presentation

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Title: Introduction to 2D NMR


1
Introduction to 2D NMR
  • Multipulse techniques

Organic Structure Analysis, Crews, Rodriguez and
Jaspars
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Organic Structure Analysis, Crews, Rodriguez and
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ONE-PULSE SEQUENCE
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ONE-PULSE SEQUENCE
(90o)x
1H
Preparation
Detection
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BASIC LAYOUT OF A 2D NMR EXPERIMENT
Preparation
Mixing t
Evolution t1
Detection t2
Organic Structure Analysis, Crews, Rodriguez and
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INVERSION-RECOVERY PULSE SEQUENCE
(180o)x
(90o)x
t2
t1
1H
Preparation
Evolution
Detection
Organic Structure Analysis, Crews, Rodriguez and
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INVERSION-RECOVERY PULSE SEQUENCE
t1
t1
Organic Structure Analysis, Crews, Rodriguez and
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SPIN-ECHO PULSE SEQUENCE
(90o)x
(180o)x
t1
t1
t2
13C
Prep.
Evolution
Detection
Organic Structure Analysis, Crews, Rodriguez and
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SPIN-ECHO PULSE SEQUENCE
FT gives null signal
Organic Structure Analysis, Crews, Rodriguez and
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10
1H-1H COSY (COrrelated SpectroscopY)
(90o)x
(90o)x
t2
t1
1H
Preparation
Evolution
Detection
Organic Structure Analysis, Crews, Rodriguez and
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PROCESSING 2D DATA
n is the number of increments
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TYPES OF 2D NMR EXPERIMENTS
  • AUTOCORRELATED
  • Homonuclear J resolved
  • 1H-1H COSY
  • TOCSY
  • NOESY
  • ROESY
  • INADEQUATE
  • CROSS-CORRELATED
  • Heteronuclear J resolved
  • 1H-13C COSY
  • HMQC
  • HSQC
  • HMBC
  • HSQC-TOCSY

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AUTOCORRELATED EXPERIMENTS 1H-1H COSY
f1f2diagonal
Gives
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AUTOCORRELATED EXPERIMENTS 1H-1H COSY
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REQUIREMENTS FOR 1H-1H COSY
  • Number of transients required is half that needed
    to give decent 1D 1H NMR spectrum
  • Most of the time we use a double quantum
    filtered COSY (DQF-COSY)
  • Same information as COSY but removes single
    quantum transitions (large singlet peaks from Me
    groups), meaning we can see things closer to the
    diagonal. Solves problems in case where there is
    a dynamic range problem (very large and very
    small peaks in same spectrum)
  • It is phase sensitive, we acquire 2 x number of
    increments (real and imaginary). Get coupling
    information from phases of correlation peaks.

Organic Structure Analysis, Crews, Rodriguez and
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PEAK PICKING FOR 1H-1H COSY
COSY
DQF-COSY
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PEAK PICKING FOR DQF-COSY
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TOtal Correlation SpectroscopY (TOCSY) HOmonuclear
HArtman-HAhn spectroscopy (HOHAHA)
Increasing the mixing time (30 180 ms)
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TOtal Correlation SpectroscopY (TOCSY) HOmonuclear
HArtman-HAhn spectroscopy (HOHAHA)
dH
dH
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TOtal Correlation SpectroscopY (TOCSY)HOmonuclear
HArtman-HAhn spectroscopy (HOHAHA)
  • Like COSY in appearance
  • Relies on relayed coherence during spin-lock
    mixing time
  • The longer tmix, the longer the correlations (30
    180 ms gives 3 - 7 bonds)
  • Relays can occur only across protonated carbons
    not across quaternary carbons (spin systems)
  • Very useful for systems containing discrete units
    eg proteins and polysaccharides

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NOESY (Nuclear Overhauser Effect
SpectroscopY) ROESY (Rotating Overhauser Effect
SpectroscopY)
Through-space correlations Up to 5 Å
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NOESY (Nuclear Overhauser Effect SpectroscopY)
dH
MW 300 Da
tmix 800 ms
dH
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ROESY (Rotating Overhauser Effect SpectroscopY)
dH
MW 800 Da
tmix 300 ms
dH
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NOESY (Nuclear Overhauser Effect
SpectroscopY)ROESY (Rotating Overhauser Effect
SpectroscopY)
  • Give through-space correlations up to 5 Å
  • The effect relies on molecular size. The NOE
    effect 0 at 1000 Da. It works well for small
    molecules (tmix 800 ms) and macromolecules
    (tmix 100 ms).
  • In the intermediate range use ROESY with tmix
    200-300 ms
  • Both NOESY and ROESY need long relaxation delays
    (2 s)
  • True NOE and ROE peaks are negative. In NOESY can
    get COSY peaks showing (positive). In ROESY can
    get TOCSY peaks showing (antiphase).
  • To determine mixing time do inversion-recovery
    experiment to find average T1. As a rule of
    thumb, NOESY tmix T1/0.7, ROESY tmix T1/1.4

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INADEQUATE Incredible Natural Abundance DoublE
QUAntum Transfer Experiment
13C-13C
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INADEQUATE Incredible Natural Abundance DoublE
QUAntum Transfer Experiment
dC
dC
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INADEQUATE Incredible Natural Abundance DoublE
QUAntum Transfer Experiment
  • C-C correlation experiment
  • Relies on two 13C being adjacent.
  • Chance of 13C-13C 1/10 000
  • Works by suppressing 13C single quantum signal
    (hence DQ)
  • Needs signal/noise of 25/1 with 1 transient 13C
    NMR experiment to get spectrum in 24 h
  • For compound of 150 Da, need 700 mg in 0.7 mL
    CDCl3 ( 6M)
  • With low volume probes and image recognition
    software can get away with much smaller samples
    and poorer signal/noise

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HETERO CORRELATED EXPERIMENTS (13C-1H)13C
DETECTED
  • 1H-13C COSY (also called HETCOR). Two types
  • Direct correlations (1JCH 140 Hz) C-H
  • Indirect (long-range) correlations (2-3JCH 9
    Hz) C-C-H and C-C-C-H
  • Very insensitive
  • For J 140 Hz take 1/3 number of transients
    needed to get 13C NMR spectrum with S/N 20/1.
    If 300 transients for 13C NMR, 2D with 256
    increments takes 14 h.
  • For J 9 Hz take 1/2 number of transients needed
    to get 13C NMR spectrum with S/N 20/1. Needs
    longer relaxation time (2s). If 300 transients
    for 13C NMR, 2D with 256 increments takes 32 h.
  • Outdated

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HETERO CORRELATED EXPERIMENTS (13C-1H)1H
(INVERSE) DETECTED
  • Direct correlations (C-H, 1JCH 140 Hz) obtained
    from HMQC or HSQC experiment (Heteronuclear
    Multiple/Single Quantum Coherence)
  • Indirect (long-range) correlations (C-C-H,
    C-C-C-H, 2-3JCH 9Hz) obtained from HMBC
    experiment (Heteronuclear Multiple Bond
    Correlation). Set JCH to other values for certain
    systems.
  • These experiments are 1H detected and have
    inherent sensitivity advantage (gH 4gC) Chance
    of 13C-1H is 1/100
  • With pulsed field gradients (PFG), it is possible
    to run 2D heterocorrelated experiments with
    single transients and 256 increments in 8-15
    minutes!
  • Without PFG need to phase cycle to remove
    artefacts. (4 transients minimum t 30 min but
    64 for full phase cycle t 9h).

Organic Structure Analysis, Crews, Rodriguez and
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HSQC versus HMQC
  • HMQC
  • Absolute value
  • Half the resolution of an HSQC
  • Can alter pulse sequence to get HMBC
  • HSQC
  • Phase sensitive
  • Double the resolution of an HMQC
  • Can edit to get positive peaks for CH, CH3 and
    negative peaks for CH2.

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HSQC Heteronuclear Single Quantum Coherence
1JCH 140 Hz C-H direct correlations (1 bond)
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HSQC Heteronuclear Single Quantum Coherence
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Edited HSQC Heteronuclear Single Quantum
Coherence
CH3
CH2
CH
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HMBC Heteronuclear Multiple Bond Correlation
2-3JCH 9 Hz C-H indirect (long range)
correlations (2-3 bonds) C-C-H C-C-C-H
Organic Structure Analysis, Crews, Rodriguez and
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HMBC Heteronuclear Multiple Bond Correlation
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3D Experiments HSQC-TOCSY
Mixing time 30-180 ms 3-7 bonds
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3D Experiments HSQC-TOCSY
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3D Experiments HSQC-TOCSY
  • 3D experiment condensed into 2D.
  • Concatenation of HSQC and TOCSY pulse sequences
  • Sorts TOCSY correlations in spin system according
    to carbon chemical shift increases resolution
    of TOCSY by adding 13C dimension
  • See direct (C-H) correlations as in HSQC, and
    long range correlations within spin systems
    depending on mixing time (30 180 ms, 3 7
    bonds). Cant go across quaternary C or
    heteroatom as it the TOCSY effect needs protons.
  • Very effective for modular systems with separate
    spin systems such as polysaccharides and peptides.

Organic Structure Analysis, Crews, Rodriguez and
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General procedure for running 2D spectra
  • Insert sample, tune 1H and 13C channels
  • Lock and shim (determine 90o pulse width)
  • Acquire 1H NMR spectrum
  • Change spectral window to 1 ppm of spectrum
  • Re-acquire 1H spectrum
  • Phase spectrum, apply baseline correction
  • Acquire 13C spectrum in optimum spectral window
  • Call up macro for 2D experiment. Use 1H and 13C
    parameters for 2D experiments
  • Alter number of transients, number of increments
    to fit the time available
  • Repeat steps 8 9 for other 2D experiments
    required
  • Set experiments running

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Processing 2D spectra Phase sensitive
experiments (DQF-COSY, TOCSY, NOESY, ROESY, HSQC,
HSQC-TOCSY)
  • Fourier transform the first increment
  • Apodise t2 using shifted sine bell squared
  • Fourier transform t2 ? f2 using apodisation
    function in 2.
  • Apodise t1 using shifted sine bell squared
  • Fourier transform t1 ? f1 using apodisation
    function in 4.
  • Phase spectrum in both dimensions if necessary

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Processing 2D spectra Absolute value
experiments (COSY, HMBC)
  • Fourier transform the first increment
  • Apodise t2 using sine bell
  • Fourier transform t2 ? f2 using apodisation
    function in 2.
  • Apodise t1 using sine bell
  • Fourier transform t1 ? f1 using apodisation
    function in 4.
  • No phasing necessary

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APODISATION - Phase sensitive experiments
APODISATION - Absolute value experiments
Organic Structure Analysis, Crews, Rodriguez and
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