Introduction to 2D NMR

Multipulse techniques

Organic Structure Analysis, Crews, Rodriguez and Jaspars

Mo
(a) No Bo

(b) Bo on; prior to resonance Net polarization Mz is due to population excess in higher energy state The magnetic vectors precess about Bo at the Larmor frequency o

Bo

y Mxy = 0

Random orientation of magnetic dipoles

(c) At resonance o = 1 The magnetic vectors precess in phase with frequency 1. After resonance the return to the equilibrium in (b) occurs by the loss of Mxy via dephasing of nuclear dipoles by T2 and increase in Mz by spin inversion due to T1.

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ONE-PULSE SEQUENCE

Bo z Mo y x
x z

Bo

M
y

(90o)x

FT

time t2
Excess of spin population along the direction of applied magnetic field.
After 90o pulse magnetization is tipped into the xy plane.

frequency f2

M=Magnetization which produces the FID. It decays as magnetization in xy plane diminishes after resonance

preparation

detection

Organic Structure Analysis, Crews, Rodriguez and Jaspars

ONE-PULSE SEQUENCE

(90o)x
1H

Preparation

Detection

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BASIC LAYOUT OF A 2D NMR EXPERIMENT

Preparation

Evolution t1

Mixing t

Detection t2

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INVERSION-RECOVERY PULSE SEQUENCE

1H

(180o)x

t1

(90o)x

t2

Preparation

Evolution

Detection

Organic Structure Analysis, Crews, Rodriguez and Jaspars

INVERSION-RECOVERY PULSE SEQUENCE

Bo z Mo
(180o)x

Bo z (90 )x y x x
o

Bo z

FT
y

y x

t1
z

Mz<0
Bo z Bo

negative (emission) peak

(90o)x y x x

FT nulled peak

Mz=0

t1
z

Bo z (90 )x y
o

Bo

FT
y x

Mz>0

positive (absorption) peak

Organic Structure Analysis, Crews, Rodriguez and Jaspars

SPIN-ECHO PULSE SEQUENCE

13C

(90o)x

t1

(180o)x

t1

t2

Prep.

Evolution

Detection

Organic Structure Analysis, Crews, Rodriguez and Jaspars

SPIN-ECHO PULSE SEQUENCE

Bo z Mo y' x'

Bo z

Bo

Bo z

-JCH/2
y'

-JCH/2
(180o)x

t=1/4JCH
y'
x'

y'

x'

+JCH/2
(180 )x at
Bo
o

for CHCl3
(90 )xat
Bo z
o

t=1/4JCH
13

at C, H +JCH/2 t=0
13

13

x'

FT gives null signal

C t=0
Bo z

-JCH/2
y'

y' x'

t=1/4JCH
y' x'

at t1=1/2JCH FT refocused

x'

+JCH/2
FT

t=1/4JCH

Organic Structure Analysis, Crews, Rodriguez and Jaspars

1H-1H

COSY (COrrelated SpectroscopY)

1H

(90o)x

t1

(90o)x

t2

Preparation

Evolution

Detection

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PROCESSING 2D DATA

t2
t1 2t1 3t1

t1
FT FT FT

f2
FT

transform matrix

FT FT

f1

nt1

FT

FT

f2

f2

t1

n is the number of increments

Organic Structure Analysis, Crews, Rodriguez and Jaspars

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars

AUTOCORRELATED EXPERIMENTS 1H-1H COSY

a f e d' d c Vicinal (3 bond) 4 bond Diagonal b a Geminal (2 bond) b c d d' e f

f1=f2=diagonal

Gives:

JHH
H H

JHH
H H

JHH
H H

allylic
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AUTOCORRELATED EXPERIMENTS 1H-1H COSY

Organic Structure Analysis, Crews, Rodriguez and Jaspars

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 Jaspars

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)

a f e d' d c Correlation Diagonal b c d d' e f

b a

Increasing the mixing time (30 180 ms):

H C

H C

H C

H C

H C

H C

Organic Structure Analysis, Crews, Rodriguez and Jaspars

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

H N N H O

H N N H O Ph

OH

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NOESY (Nuclear Overhauser Effect SpectroscopY) ROESY (Rotating Overhauser Effect SpectroscopY)
a f e d' d c Correlation (Negative) Diagonal (Positive) COSY correlation b c d d' e f

b a

Through-space correlations Up to 5
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NOESY (Nuclear Overhauser Effect SpectroscopY)

dH

MW = 300 Da tmix = 800 ms

dH
Organic Structure Analysis, Crews, Rodriguez and Jaspars

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 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

13

C axis F E D C

(ppm)

13C-13C

B A Double quantum axis (Hz)

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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

Organic Structure Analysis, Crews, Rodriguez and Jaspars

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars

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 Jaspars

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.

Organic Structure Analysis, Crews, Rodriguez and Jaspars

HSQC Heteronuclear Single Quantum Coherence

A f e d' d c B C D E F dC

b a dH

1J

CH

= 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

A f e d' d c B C D E F dC

b a dH

2-3J CH

= 9 Hz; C-H indirect (long range) correlations (2-3 bonds) C-C-H & C-C-C-H
Organic Structure Analysis, Crews, Rodriguez and Jaspars

HMBC Heteronuclear Multiple Bond Correlation

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3D Experiments HSQC-TOCSY
A f e d' d c
Direct correlations (C-H) Indirect (long range) correlations

dC

b a dH

H C

H C

H C

H C

H C

H C

H C

Mixing time 30-180 ms 3-7 bonds

Organic Structure Analysis, Crews, Rodriguez and Jaspars

3D Experiments HSQC-TOCSY

Organic Structure Analysis, Crews, Rodriguez and Jaspars

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 Jaspars

General procedure for running 2D spectra

1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars

Processing 2D spectra Phase sensitive experiments (DQF-COSY, TOCSY, NOESY, ROESY, HSQC, HSQC-TOCSY)
1. 2. 3. 4. 5. 6. 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

Organic Structure Analysis, Crews, Rodriguez and Jaspars

Processing 2D spectra Absolute value experiments (COSY, HMBC)

1. 2. 3. 4. 5. 6. 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

Organic Structure Analysis, Crews, Rodriguez and Jaspars

APODISATION - Phase sensitive experiments

Sine bell

Sine bell squared

Shifted sine bell squared

APODISATION - Absolute value experiments

Sine bell

Organic Structure Analysis, Crews, Rodriguez and Jaspars