Nuclear magnetic resonance spectroscopy: commonly referred to as NMR, is a technique which exploits the magnetic properties of certain nuclei to study physical, chemical, and biological properties of matter

NMR Spectroscopy

NMR History• 1937 Rabi’s prediction and observation of nuclear magnetic resonance

• 1945 First NMR of solution (Bloch et al for H2O) and solids (Purcell et al for parafin)!

• 1953 Overhauser NOE (nuclear Overhauser effect)

• 1966 Ernst, Anderson Fourier transform NMR

• 1975 Jeener, Ernst 2D NMR

• 1980 NMR protein structure by Wuthrich

• 1990 3D and 1H/15N/13C Triple resonance

• 1997 Ultra high field (~800 MHz) & TROSY(MW 100K)

Continuation of NMR HistoryNobel prizes

1944 Physics Rabi (Columbia)

1991 Chemistry Ernst (ETH)

"for his resonance method for recording the magnetic properties of atomic nuclei"

1952 Physics Bloch (Stanford), Purcell (Harvard)

"for their development of new methods for nuclear magnetic precision measurements and discoveries in connection therewith"

"for his contributions to the development of the methodology of high resolution nuclear magnetic resonance (NMR) spectroscopy"

Continuation of NMR History2002 Chemistry Wüthrich (ETH)

2003 Medicine Lauterbur (University of Illinois in Urbana ), Mansfield (University of Nottingham)

"for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution"

"for their discoveries concerning magnetic resonance imaging"

Basic Concepts of NMR

NMR basically a non-destructive analytical technique which is based on magnetic properties of nuclei of interest

1H NMR (Proton NMR): Gives information about types of protons and their immediate environment

13C NMR (Carbon NMR): Gives direct information about the carbon skeleton of an organic molecule

NMR stems from the same basic principle as all other forms of spectroscopy. Energy interacts with a molecule, and

absorptions occur only when the incident energy matches the energy difference between two states

The Nuclear Magnet

A spinning charge, whether positive or negative, constitutes a circular electric current which generates a magnetic dipole along the spin axis.

Since all nuclei carry charge, a spinning nucleus behaves as a tiny bar magnet, called Nuclear Magnet, placed along the spin axis, and has a characteristic magnetic moment, µ.

Nuclear SpinAn intrinsic form of angular momentum possessed by atomic 

nuclei containing an odd number of nucleons (protons or neutrons).

The Nuclear Spin is different from the electron spin. The nuclear spin represents the total angular momentum of the nucleus. It is represented by symbol, I. The nucleus is, although, composed of neutrons and protons but it acts as if it is a single entity which has intrinsic angular momentum.

The nuclear spin depends on the mass number, if the mass number is odd then the nucleus has half-integer spin like the electron while if the nucleus has even mass number then its spin will be integer spin.

Nuclear Spin Quantum Number (I)Quantum Numbers are a set of values which describes the state of any electron including the orientation and type of orbital where it is likely to be found, its spin and its distance from the nucleus.

The nuclear spin quantum number I, determines the allowed spin states of the nucleus and it is represented by;

Allowed Nuclear Spin States = 2 I + 1

In the case of hydrogen, l = 12 and hence the allowed spin states of the nucleus is 2. Similarly if the value of l of an element is known we can find the allowed states of the nucleus of that atom.

Nuclear Spin StatesThe nuclear spin states can take any number, fraction or integer. The number is dependent on the three points,

1. If both the neutrons and the protons in the nucleus are even in number then the nucleus has NO spin states.

2. If the sum of the neutrons and protons in the nucleus is odd then the nucleus has half integer spin (1/2. 3/2, 5/2, …)

3. If both the neutrons and the protons in the nucleus are odd in number then the nucleus has an integer spin states (1, 2, 3, …)

In other words the nucleus with odd number of protons or neutron or both should have the nuclear spin while if both are

even then there is no nuclear spin.

Fermions : Odd mass nuclei with an odd number of nucleons have fractional spins.

I = 1/2 ( 1H, 13C, 19F, 31P ), I = 3/2 ( 11B, 33S ) & I = 5/2 ( 17O ).

Bosons : Even mass nuclei with odd numbers of protons and neutrons have integral spins.

I = 1 ( 2H, 14N )

Even mass nuclei composed of even numbers of protons

and neutrons have zero spin

I = 0 (12C, and 16O, 32S)

Spin of Nuclei

The Nuclear Spin Resonance is study of the magnetic nuclei under the influence of the external magnetic field. The NSR is the phenomena used in many scientific instruments and experiment, in which a magnetic nucleus absorbs and emits the electromagnetic radiations under the influence of the external magnetic field.

Nuclear Spin Resonance / Spin Flipping

In the normal condition the nuclear spin is randomly oriented but under the influence of the external magnetic field the nuclear spin either aligns in the same direction with the applied magnetic field or aligns in the opposite direction with the applied magnetic field. The nuclear spin aligned with the magnetic field has lower energy than the nuclear spin aligned opposite to the magnetic field.

Energy DifferentiationIn the presence of an external magnetic field (B0), two spin states exist, +1/2 and -1/2 (For I=1/2).

The magnetic moment of the lower energy +1/2 state (α)is aligned with the external field, and that of the higher energy -1/2 ( β)spin state is opposed to the external field. The energy difference (ΔE) between these spin states is directly proportional to the strength of the applied magnetic field, Ho, and is given as:

ΔE = hγHo / 2π as ΔE = hν

In terms of frequency ν = γHo / 2π

Where γ is proportionality constant which determines sensitivity of a nucleus to the applied magnetic field and is a measure of magnetic strength of the nuclear magnet.

Magnetogyric ratio (γ)In fact, γ is the ratio between the magnetic moment (µ) and spin quantum number (I) of a nucleus, called magnetogyric ratio and is characteristic of each nucleus.

γ = 2πµ / h I

For a proton, γ has the value of 26, 764 radians / gauss . Sec

Even in a very strong magnetic field, the energy difference between the two spin states of proton is very small. For example, in a magnetic field of 23, 500 gauss (2.35 T), the energy difference (ΔE) between the spin states of proton is:

ΔE = hγHo / 2π= 6.625 x 10-34 J.s x 26764 G-1.s-1 x 23500 G / 2 x 3.143

= 6.625 x 10-26 JThis is a small amount of energy and corresponds to the radiowave

region of the electromagnetic spectrum.

Mechanism of Energy Absorption during Spin Flipping

Larmor Precession / Wobbling In addition to alignment of nuclei with a magnetic moment, application of an external magnetic field will produce a secondary spin or wobble (precession) of nuclei around the main or static magnetic field. The precessional path around the magnetic field is circular like a spinning top.

Larmor or Precessional Frequency

Spinning particle precesses about the external field axis with an angular frequency known as the Larmor frequency

wL = g Bo

When radio frequency energy matching the Larmor frequency is introduced at a right angle to the external field, it would cause a transition between the two energy levels of the spin. In other world, the precessing nucleus will absorb energy and the magnetic moment will flip to its I = _1/2 state

Precessional Frequency for Some Nuclei at 1 T

Isotope  Net Spin  g / MHz T-1 Abundance / %

1H 1/2  42.58 99.98

2H 1  6.54 0.015

3H 1/2 45.41 0.0

31P 1/2  17.25 100.0

23Na 3/2  11.27 100.0

14N 1  3.08 99.63

15N 1/2 4.31 0.37

13C 1/2  10.71 1.108

19F  1/2  40.08 100.0

Precessional Frequency of Proton at Various Magnetic Field Strengths

Precessional Frequency of 13C

wL = g Bo

The precessional frequency of a nucleus depends upon its Magnetogyric ratio (γ) and strength of applied magnetic field (Bo).

Therefore, in a magnetic field of 23, 500 gauss (2.35 T) a 1H having magnetogyric ratio (26, 764 radians G-1 S-1) precesses with a frequency of 100 MHz.

Whereas the precessional frequency of 13C in the same magnetic field will be 25.2 MHz because the magnetogyric ratio for 13C (6, 728 radians G-1 s-1) is about one-fourth of that of 1H.

Relationship between Frequency and Magnetic Field Strength

In a given sample of a specific NMR-active nucleus, the nuclei will be distributed throughout the various spin states available. As 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 number of nuclei in each spin state is described by the Boltzmann distribution :

Nupper /Nlower = e-γBo/kT

For example, at room temperature in a given sample of 1H nuclei in an external magnetic field of 1.41 Tesla the ratio between upper energy and lower energy population is only 0.9999382.

Population Distribution

Population Distribution

Effect of a radio frequency(Spin Relaxation)

Radiationless Spin RelaxationA nucleus in the higher energy spin state returns to the lower energy spin state, by losing the excess energy to some magnetic vector present in the surrounding.

Spin-Lattice relaxation (T1) / Longitudinal relaxationLattice refers to the framework of molecules which may belong to sample or solvent in any form

All these molecules have nuclei precessing therefore a variety of magnetic fields are present in lattice

A small magnetic field properly oriented in the lattice and precessing with a comparable frequency can induce transition

Spin-lattice relaxation process causes an excess of nuclei in the lower energy �spin state

Spin-lattice relaxation depends on the nature and the environment of the nucleus being observed and is useful in the structure elucidation

Spin-Spin relaxation (T2) / Transverse RelaxationSpin-spin relaxation occurs by the mutual exchange of energy by two nuclei precessing in the different spin states but the same frequency, in close proximity of each other.

It continues even in the absence of radio-frequency, individual nuclei convert from once spin state to another quite readily because they experience the magnetic field of other nearby nuclei.

Although it shortens the life-time of individual nuclei in the higher energy spin state, it does not contribute to the restoration of low energy state population excess

Radiationless Spin Relaxation

Spin Relaxation

Instrumentation of NMR

Two types of instruments are used in NMR studiesContinuous Wave (CW)The spectrometer scans through a range of frequenciesThe sample is interrogated with one frequency at a time�As the frequency range is scanned, a plot of signal intensity vs. frequency is �generated

Fourier Transform (FT)The sample is interrogated with a range of frequencies “all at once”The decay of the signal over time is observed as a Free Induction Decay (FID) PlotA Fourier transform changes the signal vs. time plot �into a signal vs. frequency plot.

Continuous Wave NMRField Sweep Mode: a constant frequency, which is continuously on, probes the energy levels while the magnetic field is varied. The energy of this frequency is represented by the blue line in the energy level diagram. 

Frequency Sweep Mode: The CW experiment can also be performed with a constant magnetic field and a frequency which is varied. The magnitude of the constant magnetic field is represented by the position of the vertical blue line in the energy level diagram.

Fourier Transform NMRIn FT-NMR, all frequencies in a spectrum are irradiated simultaneously with a radio frequency pulse. Following the pulse, the nuclei return to thermal equilibrium. A time domain emission signal is recorded by the instrument as the nuclei relax. A frequency domain spectrum is obtained by Fourier transformation.

If a signal is weak, many scans must be averaged to enhance the �signal/noise ratio.

Signal/noise ratio rises as the square root square root of number of scans Only FT instruments can obtain a large number of scans in a reasonable time.�A scan can be performed much more quickly. CW-NMR: 5 minutes; FT-NMR: 5 seconds.

Advantages of FT over CW

Components of an NMR Spectrophotometer

Powerful Magnet

Field Sweep Generator

Sample Tube

Radio-frequency Transmitter

Radio-frequency Receiver

Amplifier

Recorder

Integrator

Instrumentation of NMR

Schematic NMR Spectrometer

Instrumentation of NMRNMR Spectrophotometers are available in wide-ranging magnetic field strengths.Often described in terms of the resonance frequency of proton

Do all the protons resonate at same frequency?

Shielding of Proton

Magnetic Shielding

• If all protons absorbed the same amount of energy in a given magnetic field, not much information could be obtained.

• But protons are surrounded by electrons that shield them from the external field.

• Circulating electrons create an induced magnetic field that opposes the external magnetic field.

Shielded Protons

Magnetic field strength must be increased for a shielded proton to flip at the same frequency.

Protons in a Molecule

Depending on their chemical environment, protons in a molecule are shielded by different amounts.

=>

NMR Output

Peaks appear at the positions of absorption, also called the positions of resonance, for different nuclei in the molecule. The exact chemical shift of a particular nucleus in a molecule gives us information about how the atom with that nucleus is bonded in the molecule. The x-axis of the spectrum is called the delta scale (δ) with units of ppm and the y-axis is an intensity scale. The area under the peak on the y-axis is proportional to the number of 1H nuclei in the molecule with the same chemical shift.

Reference StandardTMS is added to the sample.Since silicon is less electronegative than carbon, TMS protons are highly shielded. Signal defined as zero.Organic protons absorb downfield (to the left) of the TMS signal. Chemically inertMiscible with most of the organic liquidsLow boiling point (27o C)All protons are magnetically equivalent

For aqueous samples

Sodium 2,2-Dimethyl- 2-Silapentane-5-Sulfonate (DSS)

The Chemical Shift

NMR absorptions generally appear as sharp peaks.

Increasing chemical shift is plotted from right to left.

Most protons absorb between 0-10 ppm.

The terms “upfield” and “downfield” describe the relative location of peaks. Upfield means to the right. Downfield means to the left.

NMR absorptions are measured relative to the position of a reference peak at 0 ppm on the delta scale due to tetramethylsilane (TMS). TMS is a volatile inert compound that gives a single peak upfield from typical NMR absorptions

The frequency of absorption for a nucleus of interest relative to the frequency of absorption of a molecular standard is called the chemical shift of the nucleus.

NMR Signals

• The number of signals shows how many different kinds of protons are present.

• The location of the signals shows how shielded or deshielded the proton is.

• The intensity of the signal shows the number of protons of that type.

• Signal splitting shows the number of protons on adjacent atoms.

Number of SignalsThe number of NMR signals equals the number of different types of protons

in a compound.

Protons in different environments give different NMR signals.

Equivalent protons give the same NMR signal.

Sample HandlingComplete homogeneity of the magnetic field Small sample ~ 1 mL of 5% sample solution in a suitable solvent3-4 cm depth in a glass tube about 15 cm long and 5 mm wideFew percent of reference standard is also added to sample solutionLiquid sample directly should not be viscousChemical shift values may have difference in different solvents (~ 1 ppm)Solvents should be mentionedNon-viscous, inexpensive, capable of dissolving sample, chemically inert, devoid of protons CCl4, CS2, C6D6, CD3CN, (CD)2SO Deuterated solvents may give a small additional peak

Sample Handling

The δ Value

The δ Value

Relative Peak Area

Molecular Structure and Chemical Shifts

•Protons in different environments experience different degrees of shielding and thus have different chemical shift values

•Shielding of a proton is determined by the structure of the molecule an is influenced by two types of intra-molecular effects1. Local diamagnetic effect / Effect of

electronegativity2. Magnetic anisotropic effect

•In fact the shielding that a proton experiences is a combination of these two types of shielding effects

Local Diamagnetic/Electronegativity Effect

•Local diamagnetic shielding is due to the circulation of electrons around the proton itself. •It therefore depends upon electron density in the immediate vicinity of the proton which in tern depends upon the electronegativity of the atom to which the proton is attached.

Methyl Halides-Within a group

Methyl Halides-Within a period

Methyl Halides- Cumulative effect

Magnetic Anisotropic Effect

Acetylene > Ethylene > Ethane

sp sp2 sp3

the directional property of the induced magnetic field of π-electrons

Therefore a proton may feel additional shielding or de-shielding depending on its orientation relative to the induced magnetic field caused by the circulation of electrons originating from the other parts of the molecule.

Such effects are called magnetic anisotropic effects.

The chemical shift values for ethane, ethylene and acetylene would increase in the same order in a regular way…

But in actual…

The local diamagnetic effects operate only along a chain of the atoms, the magnetic anisotropic effects operate through space no matter whether the influenced group is directly attached to the anisotropic group or not.

Why acetylene has a lower chemical shift value from that of ethylene?

The same phenomenon can also de-shield a proton.

4-ethynylphenanthrene; H-5 proton is 1.7 ppm downfield from…

Similar arguments can be put forward to explain the unexpectedly high chemical shift for the ethylenic protons and for the aldeydic protons.

The Ring Current Effect: Protons of the benzene ring are highly de-shielded…

Due to ring current effect the protons or any groups in the plane of the ring will be highly de-shielded while immediate above and below the ring will be highly shielded.

An interesting example of the manifestation of the ring-current effect…

The six protons inside the ring are strongly shielded (δ = -2.88 ppm) downfield from TMSWhile the twelve protons outside the ring are highly de-shielded (δ = 9.25 ppm)

The ring current effect in aromatic compounds (Cyclically delocalized π- electrons) has greater deshielding effect as compared to the conjugated alkene groups. And this criterion can be used to determine the aromatic character of an organic molecule.

δ = 2.34 ppm δ = 1.95 ppm

ortho-Hsδ = 7.85 ppm meta and para-Hs

δ = 7.40 ppm

Acetophenone

Effect of H-bondingH-bonding causes transfer of the electron-cloud from the hydrogen atom to a neighboring electronegative atom (O, N or S), resulting in deshielding of the hydrogen atom.The greater the extent of the H-bonding, the greater the deshielding of the proton and its signal will be downfield i.e. a higher chemical shift.Depends upon the type of solvent used, dilution and temperature.

Neat Ethanol (20%) in CCl4 Very dilute/Vapour5.53 ppm 2-4 ppm 0.5 ppm

Since, Intra-molecular H-bonding is not affected by dilution, temperature and solvent changes their chemical shift virtually remains constant.

Deuterium Exchange

Proton Chemical Shift Ranges

•Electronegative groups are "deshielding" and tend to move NMR signals from neighboring protons further "downfield" (to higher ppm values).•Protons on oxygen or nitrogen have highly variable chemical shifts, which are sensitive to concentration, solvent, temperature, etc.•The  -system of alkenes, aromatic compounds and carbonyls strongly deshield attached protons and move them "downfield" to higher ppm values.

Spin-Spin Coupling

Spin relaxation