nmr spectroscopy chemistry 330. 2 objectives u nmr basics u chemical shifts u relaxation times u...

Nmr Spectroscopy Nmr Spectroscopy

Chemistry 330

2

ObjectivesObjectives

Nmr basics chemical shifts relaxation times

2-Dimensional Nmr experiments COSY NOESY

What kind of information do we obtain?

3

Nmr BasicsNmr Basics

The signal in a nmr spectrum arises from transitions between nuclear spin states. 1H, 13C, 31P all have a nuclear spin quantum

number, I = 1/2. The total number of spin states

2I + 1 = 2

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Energy Levels in the Spin 1/2 Energy Levels in the Spin 1/2 SystemSystem

-1/2

+1/2

oJ

oJ

B

BhU

2

5

The Nmr SignalThe Nmr Signal

(Hz)

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The Magnetisation VectorThe Magnetisation Vector

We can represent the slight excess of spins in the state by use of the nuclear magnetization vector, Mo

x

y

z

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Shielding Shielding

The presence of the bonding electrons about the nucleus gives rise to ‘electron shields’ around the nucleus Shielding constant

)1(BB oeff

)1(2

oH B

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The Nmr Spectrum with The Nmr Spectrum with ShieldingShielding

(Hz)

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The Chemical ShiftThe Chemical Shift

Resonance frequencies are field dependent

Define a field-independent parameter - the chemical shift ()

o - magnet strength in MHz

)1(2

oH B

6

o

10 x )ppm(

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The Chemical Shift SpectrumThe Chemical Shift Spectrum

(ppm)

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J-Coupling (spin-spin splitting)J-Coupling (spin-spin splitting)

What happens when we have non-equivalent protons on adjacent C atoms?

H Ha b

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The Coupling Constant The Coupling Constant

(ppm)

Jab (Hz)

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The COSY ExperimentThe COSY Experiment

COrrelation SpectroscopY What happens when we project the

following spin system in two-dimensions?

Jab JbcHa Hb Hc

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The COSY SpectrumThe COSY Spectrum

(

ppm

)

(ppm)

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The NOESY ExperimentThe NOESY Experiment

In the 2-D NOESY (nuclear Overhauser enhancement) experiment, we look for ‘through space’ dipolar couplings.

Ha

Hb

Hc

Jab > 0

Jac = 0

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The NOESY SpectrumThe NOESY Spectrum

(

ppm

)

(ppm)

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The Origin of Spin-Lattice The Origin of Spin-Lattice RelaxationRelaxation

By using a selective pulse (a 180 pulse), we can invert the populations of the nuclear spin states

y

x

z

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Spin-Lattice RelaxationSpin-Lattice Relaxation

The spin system attempts to re-establish the equilibrium magnetisation vector. We observe the magnitude of the magnetisation vector as a function of time after the inversion pulse is applied.

(180 - n - 90 - acquire)n

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1

y

x

z

20

2

y

x

z

21

3

y

x

z

22

4

y

x

z

23

5

y

x

z

24

6

y

x

z

25

7

y

x

z

26

8

y

x

z

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Magnetisation Intensity vs. Magnetisation Intensity vs. TimeTime

-60

-40

-20

0

20

40

60

0 10 20

( arb. units)

M

(arb

. un

its)

M

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The Spin Lattice Relaxation The Spin Lattice Relaxation TimeTime

The time constant governing the decay process is the spin-lattice relaxation time, T1

M = limiting value of magnetisation intensity

M = magnetisation intensity at t =

1T

M2lnMMln

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Nuclear Overhauser Enhancement Nuclear Overhauser Enhancement (NOESY) Experiments(NOESY) Experiments

2-D Nmr NOESY experiments have been used extensively in the determination of the conformations of nucleic acids, proteins, and membranes.

The presence of the cross peak in the 2-D NOESY spectrum indicates the presence of intermolecular or intramolecular dipolar interactions (representing a spatial proximity of < 0.50 nm).

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NOESY Spectrum for NOESY Spectrum for SDS/CSDS/C44OHOH

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NOESY Spectrum for SDS/BzNOESY Spectrum for SDS/Bz

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NOESY Spectrum for NOESY Spectrum for DTAB/BzDTAB/Bz