introduction to 2d nmr multipulse techniques organic structure analysis, crews, rodriguez and...

Introduction to 2D NMR

Multipulse techniques

Organic Structure Analysis, Crews, Rodriguez and Jaspars


Random orientation of magnetic dipoles

(a) No Bo

Mo

x

yBo

Mxy = 0

(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

M z

y

x

(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.


Mo

x

y

z

Bo

Excess of spinpopulation alongthe direction ofapplied magneticfield.

(90o)x

x

y

z

Bo

After 90o pulse

magnetization

is tipped into

the xy plane.

M

time t2

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

FT

frequency f2

preparation detection

ONE-PULSE SEQUENCE


ONE-PULSE SEQUENCE

1H

(90o)x

Preparation Detection


BASIC LAYOUT OF A 2D NMR EXPERIMENT

Preparation Evolutiont1

Mixing

Detectiont2


INVERSION-RECOVERY PULSE SEQUENCE

(180o)x (90o)xt1

Preparation Evolution Detection

1Ht2


INVERSION-RECOVERY PULSE SEQUENCE

(180o)x

x

y

z

Bo

Mo

FT

Bo

z

y

x

Bo

z

y

x

(90o)x

Mz<0 negative(emission) peakt1

FT

Bo

z

y

x

(90o)x

Bo

z

y

x

Mz=0

nulled peak

t1

FT

Bo

z

y

x

Bo

z

y

x

(90o)x

Mz>0positive(absorption)peak


SPIN-ECHO PULSE SEQUENCE

(90o)x (180o)xt1

Prep. Evolution Detection

13Ct1 t2


SPIN-ECHO PULSE SEQUENCE

Mo

FT

Bo

z

y'

x'

Bo

z

y'

x'

(90o)xat 13C

for CHCl3

x'

y'

z

Bo

-JCH/2

+JCH/2

-JCH/2

t=1/4JCH

(180o)x at 13C t=0

x'

y'

z

Bo

+JCH/2

t=1/4JCH

x'

y'

z

Bo

t=1/4JCH

refocused

at t1=1/2JCH

FT

(180o)x

at 13C, 1Hx'

y'

z

Bo

-JCH/2

+JCH/2t=0

t=1/4JCH

x'

y'

z

Bo

FT gives null signal


1H-1H COSY (COrrelated SpectroscopY)

(90o)x (90o)xt1

Preparation Evolution Detection

1Ht2


PROCESSING 2D DATA

FT

FT

FT

FT

t2

t1

2t1

3t1

nt1

t1

f2

transformmatrix

t1

f2FT

FT

FT

FT f2

f1

n is the number of increments

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



AUTOCORRELATED EXPERIMENTS – 1H-1H COSY

ab

c

d

d'

ef

a b c d d' e f

Vicinal (3 bond)

Geminal (2 bond)

4 bond

Diagonal

H H H HH H

2JHH3JHH

4JHH H

H

R

H

allylic

f1=f2=diagonal

Gives:


AUTOCORRELATED EXPERIMENTS – 1H-1H COSY

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.



PEAK PICKING FOR 1H-1H COSY

COSY DQF-COSY

1

2

1

2


PEAK PICKING FOR DQF-COSY


TOtal Correlation SpectroscopY (TOCSY)HOmonuclear HArtman-HAhn spectroscopy (HOHAHA)

ab

c

d

d'

ef

a b c d d' e f

Correlation

Diagonal

Increasing the mixing time (30 – 180 ms): H

C C C C C C

H H H H H



H

H


• 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


N

N

N

N

H

OH

OH

OH

OH Ph


NOESY (Nuclear Overhauser Effect SpectroscopY)ROESY (Rotating Overhauser Effect SpectroscopY)

ab

c

d

d'

ef

a b c d d' e f

Correlation(Negative)

Diagonal(Positive)

COSY correlation

Through-space correlationsUp to 5 Å

H H


NOESY (Nuclear Overhauser Effect SpectroscopY)

H

H

MW = 300 Datmix = 800 ms


ROESY (Rotating Overhauser Effect SpectroscopY)

H

H

MW = 800 Datmix = 300 ms

• 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

NOESY (Nuclear Overhauser Effect SpectroscopY)ROESY (Rotating Overhauser Effect SpectroscopY)



INADEQUATE – Incredible Natural Abundance DoublE QUAntum Transfer Experiment

13C axis

(ppm)

Double quantum axis (Hz)

A

B

C

D

E

F13C-13C



C

C

• 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



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


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 (H = 4C) 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).


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.



HSQC – Heteronuclear Single Quantum Coherence

A B C D E F

ab

c

d

d'

ef

C

H

1JCH = 140 Hz; C-H direct correlations (1 bond)


HSQC – Heteronuclear Single Quantum Coherence


Edited HSQC – Heteronuclear Single Quantum Coherence

CH3

CH

CH2


HMBC – Heteronuclear Multiple Bond Correlation

A B C D E F

ab

c

d

d'

ef

C

H

2-3JCH = 9 Hz; C-H indirect (long range) correlations(2-3 bonds) C-C-H & C-C-C-H


HMBC – Heteronuclear Multiple Bond Correlation


3D Experiments – HSQC-TOCSY

A B C D E F

ab

c

d

d'

ef

C

H

Direct correlations (C-H)

Indirect (long range) correlations

Mixing time 30-180 ms3-7 bonds

H

C C C C C C

H H H H H

C

H


• 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). Can’t 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.


General procedure for running 2D spectra

1. Insert sample, tune 1H and 13C channels2. Lock and shim (determine 90o pulse width)3. Acquire 1H NMR spectrum4. Change spectral window to ± 1 ppm of spectrum5. Re-acquire 1H spectrum6. Phase spectrum, apply baseline correction7. Acquire 13C spectrum in optimum spectral window8. Call up macro for 2D experiment. Use 1H and 13C parameters for 2D

experiments9. Alter number of transients, number of increments to fit the time

available10. Repeat steps 8 & 9 for other 2D experiments required11. Set experiments running


Processing 2D spectra – Phase sensitive experiments (DQF-COSY, TOCSY, NOESY, ROESY, HSQC,

HSQC-TOCSY)

1. Fourier transform the first increment

2. Apodise t2 using shifted sine bell squared

3. Fourier transform t2 f2 using apodisation function in 2.

4. Apodise t1 using shifted sine bell squared


6. Phase spectrum in both dimensions if necessary


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

1. Fourier transform the first increment

2. Apodise t2 using sine bell


4. Apodise t1 using sine bell


6. No phasing necessary



Sine bell Sine bell squared Shifted sine bell squared

APODISATION - Phase sensitive experiments

APODISATION - Absolute value experiments

Sine bell

introduction to 2d nmr multipulse techniques organic structure analysis, crews, rodriguez and...

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jaspars slide

h cosy slide

h cosy cosydqfcosy slide

pulse sequence slide

h cosy f

h cosy correlated spectroscopy

cosy dqfcosy

o x preparationdetection