NMR spectroscopy

Prepared by Dr. Upali Siriwardane

For

CHEM 466 Instrumental Analysis class

Objectives

Student should gain better understanding of NMR spectroscopy.

Student should gain experience in the acquisition, processing, and displaying NMR data.

Student should gain experience in interpreting NMR data in order to establish structure for unknown organic molecules.

Student should gain understanding in advanced 1Dimensional and 2Dimensional NMR techniques.

Introduction

The Nobel Prize has been awarded twice for work related to NMR. F. Bloch and E.M. Purcell received the Nobel Prize in Physics, in 1952, for the first experimental verifications of the phenomenon, and Prof. R.R. Ernst received the Nobel Prize in Chemistry, in 1991, for the development of the NMR techniques.

Since its discovery 50 years ago, in 1945, it has spread from physics to chemistry, biosciences, material research and medical diagnosis.

The Physical Basis of the NMR Experiment

Imagine a charge travelling circularily about an axis builds up a magnetic moment

It rotates (spins) about its own axis (the blue arrow) and precesses about the axis of the magnetic field B (the red arrow). The frequency of the precession (w) is proportional to the strength of the magnetic field:

w = g B₀

= magnetogyro ratio

Magnetic field mrasured in Tesla

1 T = 10,000 gauss

Magnetogyric ratio(g)

The larger the value of the magnetogyric ratio, the larger the

Magnetic moment (m) of the nucleus and the easier it is to see by NMR spectroscopy.

Energy difference (DE) between I_z = +1/2 and I_z = -1/2.

The Physical Basis of the NMR Experiment:

Nuclear magnetic resonance, or NMR as it is abbreviated by scientists, is a phenomenon which occurs when the nuclei of certain atoms are immersed in a static strong magnetic field and exposed to a second oscillating magnetic field in the form of radiofrequency pulses, it is possible to transfer energy into the spin system and change the state of the system. After the pulse, the system relaxes back to its state of equilibrium, sending a weak signal that can be recorded.

Larmour frequency

Precession: The circular movement of the magnetic moment in the presence of the applied field.

Larmour frequency : The angular frequency of the precessionis related to the external magnetic field strength B₀, by the gyromagnetic ratio g :

w₀ = gB₀

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Quantum-mechanical treatment:

The dipole moment m of the nucleus is described in quantum-mechanical terms as

m = g J

Therein, J is the spin angular momentum and g the magnetogyric ratio of the spin. When looking at single spins we have to use a quantum-mechanical treatment.

Therein, the z-component of the angular momentum J is quantitized and can only take discrete values

J is related to spin quantum number of the nuclei I

-I,…,o,…,+I

Spin quantum number(I)

Nuclear spin is characterized by a spin number, I, which can be zero or some positive integer multiple of 1/2 (e.g. 1/2, 1, 3/2, 2 etc.). Nuclei whose spin number, I= 0 have no magnetic moment(m);eg. ¹²C and ¹⁶O show no NMR signal. Elements such as ¹H, ¹³C, ¹⁹F and ³¹P have I=1/2, while others have even higher spin numbers:

I=1 ¹⁴N, ²H

I=3/2 ¹¹B, ³⁵Cl, ³⁷Cl, ⁷⁹Br, ⁸¹Br.

As the values for I increase, energy levels and shapes of the magnetic fields become progressively more and more complex.

z-component of the angular momentum J

The energy difference DE,

Zeeman effect: splitting of energy levels in magnetic field

The energy difference DE, which corresponds to the two states with m=±1/2, is then (the quantum-mechanical selection rule states, that only transitions with m= ±1 are allowed):

A Nuclei with I= 1/2 in a Magnetic Field

A Nuclei with I= 1 in a Magnetic Field

Semi-Quantum Mechanical Approach to the Basis of NMR,

Boltzmann Distribution of Spin States

In a given sample of a specific nucleus, the nuclei will be distributed throughout the various spin states available. Because the energy separation between these states is comparatively small, energy from thermal collisions is sufficient to place many nuclei into higher energy spin states. The numbers of nuclei in each spin state are described by the Boltzman distribution

Boltzman distribution

where the N values are the numbers of nuclei in the respective spin states, is the magnetogyric ratio, h is Planck's constant, H(B) is the external magnetic field strength, k is the Boltzmann constant, and T is the temperature.

In NMR, the energy separation of the spin states is comparatively very small and while NMR is very informative it is considered to be an insensitive technique .

Example: Boltzman distribution

For example, given a sample of ¹H nuclei in an external magnetic field of 1.41 Tesla

ratio of populations = e^{((-2.67519x10e8
rad.s-1.T-1 * 1.41T * 6.626176x10-34 J.s) / (1.380662x10e-23 J.K-1 *K 293))} = 0.9999382

At room temperature, the ratio of the upper to lower energy populations is 0.9999382. In other words, the upper and lower energy spin states are almost equally populated with only a very small excess in the lower energy state.

If N₀= 10⁶ or 1,000,000then N_j 999,938

N₀- N_j =1,000,000 – 999,938 = 62

62 ppm excess in the ground state

Saturation

The condition that exists when the upper and lower energy states of nuclei are equal. (no observed signal by NMR)

Electron Spin Resonance Spectroscopy
ESR

ESR or Electron Paramagnetic Resonance (EPR) Spectroscopy

Provides information about the electronic and molecular structure of paramagnetic metal centers. Measurement of the spin state, S, the magnitude of hyperfine interactions with metal and ligand nuclei, and the zero-field splitting of half-integer S > 1/2 electronic states, allows a researcher to identify the paramagnetic center, and to potentially identify ligating atoms.

Nuclear hyperfine coupling constants

ESR Spectroscopy

Uses microwave radiation on species that contain unpaired electrons placed ina magnetic fieled

Free radicals

Odd electron molecules

Transition-metal complexes

Lanthanide ions

Triplet-state molecules

ESR of Mn²⁺

Mn²⁺is d⁵term symbol is D ( -3,-2,-1,0,+1,+2,+3) M_L= ± 1 five main spin transitions due to the D term. Hyperfine interaction each of these lines is in turn split into six components (the Mn²⁺ nuclear spin is I = 5/2) (2I+1)

Electron Spin Resonance Spectroscopy
ESR

A magnetic field splits the M_S = ±1/2 spin states into two energy levels, separated by. Because of the difference in mass of p+ and e-, a given field B will

split the electron states about 2000-fold further than the proton states.

The macroscopic view

The NMR experiment measures a largenumber of spins derived from a huge number of molecules. Therefore, we now look at the macroscopic bevaviour.

The sum of the dipole moments of all nuclei is called magnetization. In equilibrium the spins of I=1/2 nuclei are either in the a or b-state and precess about the axis of the static magnetic field. However, their phases are not correlated.

For each vector pointing in one direction of the transverse plane a corresponding vector can be found which points into the opposite direction:

Vector representation

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How NMR is achieved

Liq N₂Liq He Magnet

Instrument and Experimental Aspects

Sample Preparation,

Standards,

The probe, Probe

Tuning and Matching,

Locking, and Shimming.

Nuclear Magnetic Resonance

Sample Preparation

NMR samples are prepared and run in 5 mm glass NMR tubes. Always fill your NMR tubes to the same height with lock solvent

Deuteron resonance serves as lock- signal for the stabilisation of the spectrometer magnetic fieled.

Common NMR solvents

Acetone- d₆                    Ethanole- d₆                   Acetonitrile- d₃

Formic acid- d₂               Benzene- d₆                  Methanole- d₄

Chloroform- d₁                Nitromethane- d₃         Deuteriumoxide-D₂O

Pyridine- d₅                     Dichloromethane- d₂   1,1,2,2- Tetrachloroethane- d₂ Dimethylformamide- d₇ Tetrahydrofurane- d₈    Dimethylsulfoxide- d₆

Toluene- d₈                      1,4- Dioxane- d₈           Trifluoroacetic acid- d₁

NMR solvents are used as reference peaks

to adjust the ppm values in the spectrum

relative to TMS (tetramethyl silane)

NMR probes

NMR probes designed creating different radio frequency singnals and detectors for dealing with varuous magnetic nuclie have become more advanced and allow progressively smaller samples. Probe diameters and correspondingly sample volumes have progressively decreased.

¹H NMR Probe High frequency ( 270 MHz)probes

¹⁹F NMR Probe High frequency (254 MHz) probes

¹³C NMR Probe Low frequncy(< 254 MHz) probes

Broad band probe High/Low frequency tunable probes

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Shielding and Deshielding of Nuclei

The magnetic field at the nucleus, B, (the effective field) is therefore generally less than the applied field, B_o, by a fraction .

B = B_o (1-s)

peaks move to right due to shileding

peaks move to left due to deshileding: beeing attached more electronegitve atoms or experiencing ring currents as in benezne

Chemical Shift

The chemical shift of a nucleus is the difference between the resonance frequency of the nucleus and a standard, relative to the

standard. This quantity is reported in ppm and given the symbol delta, d.

d = (n - n_REF) x10⁶ / n_REF

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Spin-Spin Coupling

Nuclei which are close to one another exert an influence on each other's effective magnetic field. This effect shows up in the NMR spectrum when the nuclei are nonequivalent. If the distance between non-equivalent nuclei is less than or equal to three bond lengths, this effect is observable. This effect is called spin-spin coupling or J coupling.

Spin-Spin Coupling

For the next example, consider a molecule with spin 1/2 nuclei, one type A and type B

This series is called Pascal's triangle and can be calculated from the coefficients of the expansion of the equation (x+1)ⁿ

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The types of information accessible via high resolution NMR include

1.Functional group analysis (chemical shifts)

2.Bonding connectivity and orientation (J coupling)

3.Through space connectivity (Overhauser effect)

4.Molecular Conformations, DNA, peptide and enzyme sequence and structure.

5.Chemical dynamics (Lineshapes, relaxation phenomena).

Multinuclear NMR

Spin angular momentum number of I =1/2, of which examples are ¹H, ¹³C, ¹⁵N, ¹⁹F, ³¹P

How NMR Signals are Created, Relaxation

FT-NMR Experimental Method

Data Acquisition and Storage,

Digital Resolution,

Folding,

Quadrature Phase Detection.

Data Treatment

Apodization or Window Functions,

Zero Filling,

Fourier Transformation,

Phase Correction.

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Fourier Transformation

Fourier Transformation- FT

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The Proton NMR

Stereochemical Equivalent/Non-equivalent Protons

Chemical Shift

Spin Coupling

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Simplification of proton NMR Spectra

:Spin Decoupling,

Higher Field NMR Spectra,

Lanthanide Shift Reagents.

Carbon NMR Spectroscopy

Introduction,

Chemical Shifts,

Experimental Aspects of 13C NMR Spectroscopy.

2D NMR

Experimental Aspects of 2D NMR Spectroscopy.

Preparation, Evolution and Mixing,

Data Acquisition,

Spectra Presentation.

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2D Homonuclear Correlated NMR Experiments

COSY (Correlation Spectroscopy )

NOESY(NOE Nuclear Overhauser effect Spectroscopy)

TOCSY experiment correlates all protons of a spin system

ROESY- NOE in the Rotating Frame

HETCOR -heteronuclear correlation spectroscopy

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Hetero- 2D Nuclear Correlated NMR Experiments

HETCOR

HMBC

HMQC.

Magnetic Resonance Imaging (MRI)

Another growing field of interest in NMR is MR-imaging. The water content of the human body allows the making of proton charts or images of the whole body or certain tissues. Since static magnetic fields or radiopulses have been found not to injure living organisms, MR-imaging is competing with x-ray tomography as the main diagnostic tool in medicine. The MR-imaging technique has been applied to material research as well.

Magnetic Resonance Imaging
(MRI)

Functional Nuclear magnetic resonance(FMRI)

patient is placed in a tube with magnetic fields The way the ¹H in body responds to those fields is noted and sent to a computer along with information about where the interactions occurred. Myriads of these points are sampled and fed into a computer that processes the information and creates an image.

Thoughts Image Mapping by Functional Nuclear magnetic resonance FMRI


	Prepared by Dr. Upali Siriwardane
	For
	CHEM 466 Instrumental Analysis class


	Student should gain better understanding of NMR spectroscopy.
	Student should gain experience in the acquisition, processing, and displaying NMR data.
	Student should gain experience in interpreting NMR data in order to establish structure for unknown organic molecules.
	Student should gain understanding in advanced 1Dimensional and 2Dimensional NMR techniques.


	The Nobel Prize has been awarded twice for work related to NMR. F. Bloch and E.M. Purcell received the Nobel Prize in Physics, in 1952, for the first experimental verifications of the phenomenon, and Prof. R.R. Ernst received the Nobel Prize in Chemistry, in 1991, for the development of the NMR techniques.
	Since its discovery 50 years ago, in 1945, it has spread from physics to chemistry, biosciences, material research and medical diagnosis.


	Imagine a charge travelling circularily about an axis builds up a magnetic moment
	It rotates (spins) about its own axis (the blue arrow) and precesses about the axis of the magnetic field B (the red arrow). The frequency of the precession (w) is proportional to the strength of the magnetic field:
	w = g B₀
	= magnetogyro ratio
	Magnetic field mrasured in Tesla
	1 T = 10,000 gauss


	The larger the value of the magnetogyric ratio, the larger the
	Magnetic moment (m) of the nucleus and the easier it is to see by NMR spectroscopy.
	Energy difference (DE) between I_z = +1/2 and I_z = -1/2.


	Nuclear magnetic resonance, or NMR as it is abbreviated by scientists, is a phenomenon which occurs when the nuclei of certain atoms are immersed in a static strong magnetic field and exposed to a second oscillating magnetic field in the form of radiofrequency pulses, it is possible to transfer energy into the spin system and change the state of the system. After the pulse, the system relaxes back to its state of equilibrium, sending a weak signal that can be recorded.


	Precession: The circular movement of the magnetic moment in the presence of the applied field.
	Larmour frequency : The angular frequency of the precessionis related to the external magnetic field strength B₀, by the gyromagnetic ratio g :
	w₀ = gB₀


	The dipole moment m of the nucleus is described in quantum-mechanical terms as
	m = g J
	Therein, J is the spin angular momentum and g the magnetogyric ratio of the spin. When looking at single spins we have to use a quantum-mechanical treatment.
	Therein, the z-component of the angular momentum J is quantitized and can only take discrete values


	J is related to spin quantum number of the nuclei I
	-I,…,o,…,+I


	Nuclear spin is characterized by a spin number, I, which can be zero or some positive integer multiple of 1/2 (e.g. 1/2, 1, 3/2, 2 etc.). Nuclei whose spin number, I= 0 have no magnetic moment(m);eg. ¹²C and ¹⁶O show no NMR signal. Elements such as ¹H, ¹³C, ¹⁹F and ³¹P have I=1/2, while others have even higher spin numbers:
	I=1 ¹⁴N, ²H
	I=3/2 ¹¹B, ³⁵Cl, ³⁷Cl, ⁷⁹Br, ⁸¹Br.
	As the values for I increase, energy levels and shapes of the magnetic fields become progressively more and more complex.


	Zeeman effect: splitting of energy levels in magnetic field
	The energy difference DE, which corresponds to the two states with m=±1/2, is then (the quantum-mechanical selection rule states, that only transitions with m= ±1 are allowed):


	In a given sample of a specific nucleus, the nuclei will be distributed throughout the various spin states available. Because the energy separation between these states is comparatively small, energy from thermal collisions is sufficient to place many nuclei into higher energy spin states. The numbers of nuclei in each spin state are described by the Boltzman distribution


	where the N values are the numbers of nuclei in the respective spin states, is the magnetogyric ratio, h is Planck's constant, H(B) is the external magnetic field strength, k is the Boltzmann constant, and T is the temperature.
	In NMR, the energy separation of the spin states is comparatively very small and while NMR is very informative it is considered to be an insensitive technique .


	For example, given a sample of ¹H nuclei in an external magnetic field of 1.41 Tesla
	ratio of populations = e^{((-2.67519x10e8 rad.s-1.T-1 * 1.41T * 6.626176x10-34 J.s) / (1.380662x10e-23 J.K-1 *K 293))} = 0.9999382
	At room temperature, the ratio of the upper to lower energy populations is 0.9999382. In other words, the upper and lower energy spin states are almost equally populated with only a very small excess in the lower energy state.
	If N₀= 10⁶ or 1,000,000then N_j 999,938
	N₀- N_j =1,000,000 – 999,938 = 62
	62 ppm excess in the ground state


	The condition that exists when the upper and lower energy states of nuclei are equal. (no observed signal by NMR)


	ESR or Electron Paramagnetic Resonance (EPR) Spectroscopy
	Provides information about the electronic and molecular structure of paramagnetic metal centers. Measurement of the spin state, S, the magnitude of hyperfine interactions with metal and ligand nuclei, and the zero-field splitting of half-integer S > 1/2 electronic states, allows a researcher to identify the paramagnetic center, and to potentially identify ligating atoms.
	Nuclear hyperfine coupling constants


	Uses microwave radiation on species that contain unpaired electrons placed ina magnetic fieled
	Free radicals
	Odd electron molecules
	Transition-metal complexes
	Lanthanide ions
	Triplet-state molecules


	Mn²⁺is d⁵term symbol is D ( -3,-2,-1,0,+1,+2,+3) M_L= ± 1 five main spin transitions due to the D term. Hyperfine interaction each of these lines is in turn split into six components (the Mn²⁺ nuclear spin is I = 5/2) (2I+1)


	A magnetic field splits the M_S = ±1/2 spin states into two energy levels, separated by. Because of the difference in mass of p+ and e-, a given field B will
	split the electron states about 2000-fold further than the proton states.


	The NMR experiment measures a largenumber of spins derived from a huge number of molecules. Therefore, we now look at the macroscopic bevaviour.
	The sum of the dipole moments of all nuclei is called magnetization. In equilibrium the spins of I=1/2 nuclei are either in the a or b-state and precess about the axis of the static magnetic field. However, their phases are not correlated.
	For each vector pointing in one direction of the transverse plane a corresponding vector can be found which points into the opposite direction:


	Sample Preparation,
	Standards,
	The probe, Probe
	Tuning and Matching,
	Locking, and Shimming.


	Sample Preparation
	NMR samples are prepared and run in 5 mm glass NMR tubes. Always fill your NMR tubes to the same height with lock solvent
	Deuteron resonance serves as lock- signal for the stabilisation of the spectrometer magnetic fieled.


	Acetone- d₆ Ethanole- d₆ Acetonitrile- d₃
	Formic acid- d₂ Benzene- d₆ Methanole- d₄
	Chloroform- d₁ Nitromethane- d₃ Deuteriumoxide-D₂O
	Pyridine- d₅ Dichloromethane- d₂ 1,1,2,2- Tetrachloroethane- d₂ Dimethylformamide- d₇ Tetrahydrofurane- d₈ Dimethylsulfoxide- d₆
	Toluene- d₈ 1,4- Dioxane- d₈ Trifluoroacetic acid- d₁
	NMR solvents are used as reference peaks
	to adjust the ppm values in the spectrum
	relative to TMS (tetramethyl silane)


	NMR probes designed creating different radio frequency singnals and detectors for dealing with varuous magnetic nuclie have become more advanced and allow progressively smaller samples. Probe diameters and correspondingly sample volumes have progressively decreased.
		¹H NMR Probe High frequency ( 270 MHz)probes
		¹⁹F NMR Probe High frequency (254 MHz) probes
		¹³C NMR Probe Low frequncy(< 254 MHz) probes
		Broad band probe High/Low frequency tunable probes


	The magnetic field at the nucleus, B, (the effective field) is therefore generally less than the applied field, B_o, by a fraction .
	B = B_o (1-s)
	peaks move to right due to shileding
	peaks move to left due to deshileding: beeing attached more electronegitve atoms or experiencing ring currents as in benezne


	The chemical shift of a nucleus is the difference between the resonance frequency of the nucleus and a standard, relative to the
	standard. This quantity is reported in ppm and given the symbol delta, d.
	d = (n - n_REF) x10⁶ / n_REF


	Nuclei which are close to one another exert an influence on each other's effective magnetic field. This effect shows up in the NMR spectrum when the nuclei are nonequivalent. If the distance between non-equivalent nuclei is less than or equal to three bond lengths, this effect is observable. This effect is called spin-spin coupling or J coupling.


	For the next example, consider a molecule with spin 1/2 nuclei, one type A and type B








	This series is called Pascal's triangle and can be calculated from the coefficients of the expansion of the equation (x+1)ⁿ


	1.Functional group analysis (chemical shifts)
	2.Bonding connectivity and orientation (J coupling)
	3.Through space connectivity (Overhauser effect)
	4.Molecular Conformations, DNA, peptide and enzyme sequence and structure.
	5.Chemical dynamics (Lineshapes, relaxation phenomena).


	Spin angular momentum number of I =1/2, of which examples are ¹H, ¹³C, ¹⁵N, ¹⁹F, ³¹P


	Data Acquisition and Storage,
	Digital Resolution,
	Folding,
	Quadrature Phase Detection.