2D Lecture

7/28/2019 2D Lecture

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Introduction to 2D NMR

Multipulse techniques

Organic Structure Analysis, Crews, Rodriguez and Jaspars


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Randomorientation ofmagnetic dipoles

(a) NoBo

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 vectorsprecess about Bo at

the Larmor frequency o

Mz

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 ofMxy via

dephasing of nuclear

dipoles by T2 and increase

in Mz by spin inversion

due to T1.


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Mo

x

y

z

Bo

Excess of spin

population along

the direction of

applied magnetic

field.

(90o)x

x

y

z

Bo

After 90opulse

magnetization

is tipped into

thexyplane.

M

time t2

M=Magnetization which

produces the FID. It decays

as magnetization inxy

plane diminishes after

resonance

FT

frequency f2

preparation detection

ONE-PULSE SEQUENCE


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INVERSION-RECOVERY PULSE SEQUENCE

(180o)x

(90o)x

t1

Preparation Evolution Detection

1H

t2


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INVERSION-RECOVERY PULSE SEQUENCE

(180

o

)x

x

y

z

Bo

Mo

FT

Bo

z

y

x

Bo

z

y

x

(90

o

)x

Mz0

positive

(absorption)

peak


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SPIN-ECHO PULSE SEQUENCE

(90o)x

(180o)x

t1

Prep. Evolution Detection

13Ct1 t2


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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=1/2JCHFT

(180o)x

at13

C,1H

x'

y'

z

Bo

-JCH/2

+JCH/2t=0

t=1/4JCH

x'

y'

z

Bo

FT givesnull signal


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PROCESSING 2D DATA

FT

FT

FT

FT

t2

t1

2t1

3t1

nt1

t1

f2

transform

matrix

t1

f2FT

FT

FT

FT f2

f1

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 EXPERIMENTS1H-1H COSY

a

b

c

d

d'

e

f

a b c d d' e f

Vicinal (3 bond)

Geminal (2 bond)

4 bond

Diagonal

H H H HH H

2JHH 3JHH 4JHH H

H

R

H

allylic

f1=f2=diagonal

Gives:


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AUTOCORRELATED EXPERIMENTS1H-1H COSY


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REQUIREMENTS FOR 1H-1H COSY

Number of transients required is half that needed to give decent1D 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 thingscloser to the diagonal. Solves problems in case where there is adynamic range problem (very large and very small peaks in samespectrum)

It is phase sensitive, we acquire 2 x number of increments (real andimaginary). Get coupling information from phases of correlationpeaks.



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PEAK PICKING FOR 1H-1H COSY

COSY DQF-COSY

1

2

1

2


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

a

b

c

d

d'

e

f

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


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dH

dH


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Like COSY in appearance

Relies on relayed coherenceduring spin-lock mixing time

The longer tmix

, the longer thecorrelations (30 180 ms gives3 - 7 bonds)

Relays can occur only acrossprotonated carbons not acrossquaternary carbons (spin

systems) Very useful for systems

containing discrete units egproteins and polysaccharides


N

N

N

N

H

OH

OH

OH

OH Ph


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

a

b

c

d

d'

e

f

a b c d d' e f

Correlation

(Negative)

Diagonal

(Positive)COSY

correlation

Through-space correlationsUp to 5

H H


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NOESY (Nuclear Overhauser Effect SpectroscopY)

dH

dH

MW = 300 Da

tmix = 800 ms


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ROESY (Rotating Overhauser Effect SpectroscopY)

dH

dH

MW = 800 Datmix = 300 ms


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Give through-space correlations up to 5

The effect relies on molecular size. The NOE effect ~ 0 at 1000Da. It works well for small molecules (tmix ~ 800 ms) andmacromolecules (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 getCOSY peaks showing (positive). In ROESY can get TOCSYpeaks showing (antiphase).

To determine mixing time do inversion-recovery experiment tofind 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)



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INADEQUATE Incredible Natural Abundance DoublEQUAntum Transfer Experiment

dC

dC


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C-C correlation experiment

Relies on two 13C being adjacent.

Chance of13C-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 getaway with much smaller samples and poorer signal/noise

INADEQUATE Incredible Natural Abundance DoublEQUAntum Transfer Experiment



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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

ForJ= 140 Hz take 1/3 number of transients needed to get 13CNMR spectrum with S/N = 20/1. If 300 transients for13C NMR,2D with 256 increments takes 14 h.

ForJ= 9 Hz take 1/2 number of transients needed to get 13CNMR spectrum with S/N = 20/1. Needs longer relaxation time

(2s). If 300 transients for

13

C NMR, 2D with 256 incrementstakes 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 HSQCexperiment (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 BondCorrelation). Set JCH to other values for certain systems.

These experiments are 1H detected and have inherent sensitivityadvantage (gH = 4gC) Chance of

13C-1H is 1/100

With pulsed field gradients (PFG), it is possible to run 2Dheterocorrelated experiments with single transients and 256 incrementsin 8-15 minutes!

Without PFG need to phase cycle to remove artefacts. (4 transientsminimum: t = 30 min; but 64 for full phase cycle: t = 9h).



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HSQC Heteronuclear Single Quantum Coherence

A B C D E F

a

b

c

d

d'

e

f

dC

dH

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

CH

CH2


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HMBC Heteronuclear Multiple Bond Correlation

A B C D E F

a

b

c

d

d'

e

f

dC

dH

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


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3D Experiments HSQC-TOCSY

A B C D E F

a

b

c

d

d'

e

f

dC

dH

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


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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 adding13

Cdimension

See direct (C-H) correlations as in HSQC, and long rangecorrelations within spin systems depending on mixing time (30180 ms, 37 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.



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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 spectrum

5. 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

experiments

9. Alter number of transients, number of increments to fit the timeavailable10. Repeat steps 8 & 9 for other 2D experiments required11. Set experiments running



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

1. Fourier transform the first increment

2. Apodise t2 using sine bell

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

4. Apodise t1 using sine bell5. Fourier transform t1 f1 using apodisation function in 4.

6. No phasing necessary



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