NMR SPECTROSCOPY

WORKSHOP

(24- 27 January 2013)

By NMR Research Centre, IISc , Bangalore.

JEESU GEORGE

MS- 2012/05

IIRBS, MGU.

Institute for Intensive Research in Basic Sciences, MG university conducted a workshop

on “NMR spectroscopy” in associate with NMR research centre of Indian Institute of Science

from 24th to 27th of January, 2012.

PROF. K. V. RAMANATHAN

The lectures of workshop where initiated by Prof. K.V.Ramanathan from NMR Research

Centre,Indian Institute of Science Bangalore . He talked about the history and development

of N.M.R referring to the major contributions of scientists from various fields (physics,

chemistry etc.) at various times. This included the first detection of N.M.R, MS NMR, FT

NMR, 2D NMR, 2D-NMR and protein structure, development of M.R.I. etc. He then started

to tell about the contributions of many scientists into the field. These included Pauli’s

prediction of nuclear spin (1926) detection of nuclear magnetic moment by Stern (1932),

first theoretical prediction of NMR by Gorter (1936- but failure) , first NMR of solutions and

solids and discovery of chemical shifts by Bloch (1945,49), high resolution solid state NMR

by magic angle spinning (1958) , first pulse FT NMR by Ernest and Anderson (1946) etc. After

a long duration 0f 25 years it continued by proposal of 2 pulse 2D experiment by Jeener

(1971), MRI experiment by Lauterbur (1972), first demonstration of 2D NMR by Ernst

(1974), 2D 1H NOESY (1979), elucidation of protein structure by Wuthrich using NMR

(1980), triple resonance NMR of protein (1990), ultra high field and TROSY (1997), structural

determination of proteins in solids (2002) etc.

Between these slides he also emphasized about the nobel laureates in he contribution

of NMR. They are Otto Stern , Isidor I. Rabi, Felix Bloch, Edward M. Purcell, Nicolaas

Bloembergen from the fields of physics ( 1943- 1981); Richard Ernst and Kurt Wütrich from

the fields of chemistry (1991, 2002); Paul C. Lauterbur and Sir Peter Mansfield (2003). He

also didn’t forget to talk about the contributions of Indian scientists in this field. He

mentioned about G. Suryan, IISc, S.S.Dharmati, TIFR, C.L.KhetrapalL, IISc, Anil Kumar, IISc

et al from India.

After this brief history, he directly went to the science of NMR from the fundamentals.

For the observance of nuclear magnetic resonance, the nuclear spin quantum number of an

atom should be non-zero. For atoms with even atomic mass & number l=0, those with even

atomic mass & odd number, l will be a whole integer and those with odd atomic mass have

l with a half integer. Nuclear net spin considers only the unpaired particles inside the

nucleus (unpaired protons, neutrons). This nuclear spin behaves like a tiny magnet. It also

has a magnetic moment. Spin angular momentum and magnetic moment are related to

each other as µ= Ƴ I h /2π. By applying a magnetic field to an atom, the electrons

shift either to the ground state or excited state. There is an energy difference ΔE between

these states. Due to the magnetic field the particles align either parallel or anti-parallel to it.

By supplying electromagnetic energy with frequency v, we can alter these alignment and it

turns out that v is the same as the Larmor Precession Frequency given by, v = Ƴ Bo / 2 π. This

energy separation is directly proportional to magnetic field and also the magnetic moment

of the nuclei. As higher the magnetic field and larger the magnetic moment, the higher will

be the Resonance Frequency.

A tiny magnet,(compass needle) always aligns itself parallel to an external magnetic field

. Similarly, the nuclear spins also align in a magnetic field. The magnetic moment moves in a

cone keeping an angle with the magnetic field. The net magnetization of nuclear spin is

along the Z direction alone, as the XY components cancels out. The detection of these

signals is by Fourier transform method. Usually, we are applying radio frequency along Y

direction and magnetization along Z direction to get the signal along X direction. The

irradiation energy we provide is generally in the RF range and is typically applied as a short

pulse. The absorption of energy by the nuclear spins causes transitions from higher to lower

energy as well as from lower to higher energy. This two-way flipping is a hallmark of the

resonance process. The energy absorbed by the nuclear spins induces a voltage that can be

detected by a suitably tuned coil of wire, amplified, and the signal displayed as a Free

Induction Decay (FID). The FID graph is then transformed into readable signals by the

mathematical methods of Fourier.

Dr. Ramanathan then started to talk about parts of an NMR spectrometer. This

included,

A magnetic field ( intense, homogenous and stable).

A probe (enables the coil to excite and detect the signal)

Electronic circuits for radio frequency

A computer (overall control, signal processing and interpretation)

The high magnetic field has several advantages. It assists high chemical shift dispersion

which improves selectivity of spectral editing schemes. Magnetic field also supports the

high sensitivity, simplification or first order spectra, TROSY effect , field induced molecular

orientation, residual dipolar couplings etc. Modern NMR spectrometers make use of

superconducting magnets (air core) in persistent current mode. Thin wires of NbTi or

Nb3Sn alloys are used as super conducting material as they have zero resistance at very

low temperatures. The low temperature condition can be created by immersing the

superconductor in a bath of liquid helium. We use nitrogen to minimize the evaporation of

liquid helium. But, moreover the sample is always maintained to keep at room temperature.

Shim coils are used to maintain homogenous fields. Each shim coil will produce a

counteracting tiny magnetic field with a particular spatial profile so as to cancel the residual

field in homogeneities.

We obtain certain parameters from the NMR. They are: chemical shift, J coupling

constant, Nuclear Overhauser Enhancement (NOE), Relaxation time T1, Relaxation time T2,

Dipolar & Quadrupolar Coulings (Solids/Condensed matter). The NMR spectra we obtain

are influenced by the following properties: valency & bonding of the atom, geometry &

sterio-chemistry of the molecule, basicity & interaction with other molecules and isomerism

& chemical exchange.

The nuclei of different elements, having different gyro-magnetic ratios, will yield signals

at different frequencies in a particular magnetic field. However, it also turns out that nuclei

of the same type can achieve the resonance condition at different frequencies. This can

occur if the local magnetic field experienced by a nucleus is slightly different from that of

another similar nucleus. If a molecule containing the nucleus of interest is put in a magnetic

field B, simple electromagnetic theory indicates that the B field will induce electron currents

in the molecule in the plane perpendicular to the applied magnetic field. These induced

currents will then produce a small magnetic field opposed to the applied field that acts to

partially cancel the applied field, thus shielding the nucleus. In general, the induced

opposing field is about a million times smaller than the applied field. Consequently, the

magnetic field perceived by the nucleus will be very slightly altered from the applied field.

There will be more electronic currents induced in the molecule than just those directly

around the nucleus. In fact, some of those currents may increase Blocal. Therefore, the

shielding and the resulting resonance frequency will depend on the exact characteristics of

the electronic environment around the nucleus. The induced magnetic fields are typically a

million times smaller than the applied magnetic field. So if the Larmor resonance frequency

no is on the order of several megahertz, differences in resonance frequencies for two

different hydrogen nuclei, for example, will be on the order of several hertz. Although we

cannot easily determine absolute radiofrequencies to an accuracy of ±1 Hz, we can

determine the relative positions of two signals in the NMR spectrum with even greater

accuracy. Consequently, a reference signal is chosen, and the difference between the

position of the signal of interest and that of the reference is termed the chemical shift.

A nucleus with a magnetic moment may interact with other nuclear spins resulting in

mutual splitting of the NMR signal from each nucleus into multiplets. The number of

components into which a signal is split is 2nI+1, where I is the spin quantum number and n

is the number of other nuclei interacting with the nucleus. The difference between any two

adjacent components of a multiplet is the same and yields the value of the spin-spin

coupling constant J. One important feature of spin-spin splitting is that it is independent of

magnetic field strength. So increasing the magnetic field strength will increase the chemical

shift difference between two peaks in hertz (not parts per million), but the coupling

constant J will not change. After these talks Prof. Ramanathan concluded the class.

He continued with a new topic the very next day. This day he dealt with two

dimensional NMR, solid state NMR etc. two dimensional NMR has two time domains. T1 is

the variable delay time and T2 is the normal acquisition time. The signal along the T2 domain

is periodic. The domain signal along T1 can also become periodic by building pseudo FID by

looking at the points for each frequency along T2. The transformation of these FIDs by

Fourier transformation twice into the signals provides frequencies in two dimensions. We

use the two dimensional NMR in several spectroscopies like COSY(COrrelated SpectroscopY)

, TOCSY (TOtal Correlated SpectroscopY), NOESY (Nuclear Overhauser Effect SpectroscopY)

etc. COSY is the correlated spectroscopy giving the spectrum of two nearly bonded atoms.

TOCSY is the total correlation spectroscopy which is an extension of COSY where one spim

correlates to all others in the coupled spin system. It is very useful to identify different

amino acids as each amino acid is an isolated spin system. Apart from these, NOESY is the

spectroscopy through space.

He then talked about the developments in solid state NMR. As we are able to apply the

NMR spectroscopy in liquid phase we are also able to use them in solid state. But this case is

least advised that solid state NMR is used for only those compounds which are not soluble

in no solvents at all. This have low sensitivity, long T1, broad lines etc. Moreover solid state

NMR is more noisy. The magnetic field applied to the solid sample will not be equally

experienced by all the molecules equally. In order to account for these we rotate the

sample in particular angles called magic angle spinning.

Prof. Ramanathan later extended his talk to the application of NMR in various fields

including that of medicine. In hospitals we use the MRI scans which is another type of NMR.

MRI scanning machine takes the picture of the human body. The factors affecting this

scanning are the number of protons in the tissue at a particular place and the relaxation

times of the protons. In the body, the hydrogens of water attached to the surface of

biological molecules relax than those in the free fluid. Depending on the nature of the tissue

or structure to which the water is bound, there are minor differences in them. Generally

brain tumour is detected by MRI. Prof. Ramanathan gave an end to his lectures by these.

PROF. N. SURYAPRAKASH

It was Prof. N. Suryaprakash, an eminent scientist from NMR research centre of Indian

Institute, Bangalore who gave the theoretical explanations behind NMR. He started by

teaching resonance conditions in NMR. When the applied magnetic field frequency is same

as that of the precession frequency of the spins, resonance condition is induced. By applying

a magnetic field to an atom, the electrons shift either to the ground state or excited state.

There is an energy difference ΔE between these states. When energy is absorbed by the

spins when the frequency matches with the energy separation, transition occurs.

The transitions depend on the quantum numbers which are characteristics of each state

or energy level. Only transitions in which the total spin angular momentum changes by one

(up or down) are allowed. By tradition in NMR the energy level (or state) with m = ½ is

denoted α and is sometimes described as “spin up”. The state with m = - ½ is denoted β

and is sometimes described as “spin down”. There is variations in the case of energy levels

for homo and hetero- nuclear species. The α β and β α states have same energy in homo-

nuclear case. But for for hetero-nuclear case this will be different. He also told about the

factors affecting NMR spectra. They are two types : static( Chemical Shifts, Spin-spin

couplings, Dipolar Couplings& Quadrupolar couplings) and dynamic (Spin Lattice Relaxation

(T1) & Spin-Spin Relaxation (T2)).

Chemical shift is due to the magnetic interaction of the nuclear spins with external

magnetic field through electrons. External field induces currents in the electron clouds in

the molecule but the circulating molecular current in turn can generate a magnetic field.

This induced field by the electrons can oppose the magnetic field which is provided

externally. By this way the electrons can shield the protons. Various functional show

different shielding properties giving rise to different resonating frequencies.

The molecule we are providing must want to be immersed in certain solvents for the

measurement of chemical shift. Generally deuterium solvents are used as it resonates

outside the proton region and the solvent signals are reduced in intensity. Another aspect in

the NMR analysis is the type of reference we are using to measure relative values.

Generally, we are using tetramethylsilane (TMS) for this purpose. The qualities of TMS to be

used a reference are:

Volatility

Inert nature ( no molecular interactions)

Resonating at high magnetic fields.

Provides a single peak for protons and carbons.

But we will be using different references for different nuclei.

Chemical shift is an important property of molecules such that we can call it as the

finger print of molecules. Chemical shifts can vary for different functional groups, different

nuclei and for different magnetic fields.

The concept of chemical equivalence comes into account in context of chemical shifts.

Apart from different molecular structures different protons in the same environment will

have the same chemical shifts. These protons with the same chemical shifts are referred to

as chemically equivalent. Symmetry is also important in deciding the chemically equivalent

protons.

Shielding effect is another important concept we have to take into consideration.

Valence electrons density can shield nucleus from applied field. Electronegative substituent

can draw electron density away, results in deshielding . By Lamb’s formula, shielding

constant

By breaking the equation into different terms, we can understand most of the

effects. By knowing the ρ(r) value, we can use the same formula for other molecules

also. Higher the electronegativity, more the deshielding and resonating frequency is

higher (lower field). Inherently, all chemical bonds are anisotropic with all of them

having directions in space and depending on the direction we are looking at, it will be

different. The bonds may have an induced magnetic moment in an external magnetic

field which also will be anisotropic. Another important aspect is the mesomeric effect.

It may differ for electron donating groups and electron withdrawing group.

There is no distinctive scale for the exchangeable protons since the resonance positions

of these protons are strongly dependent on the medium and temperature. The formation

of hydrogen bond leads to significant down field shifts. The electrical dipole field of the

hydrogen bond, that is formulated as a pure electrostatic attractive bond appears to effect

a deshielding. Intra and intermolecular hydrogen bonding can easily be distinguished. Thus

chemical shifts through hydrogen bonding is an important aspect in NMR spectroscopy.

Shoolery rules are rules in spectroscopy for predicting the chemical shifts. If we can

estimate the different effects like electronegativity , electronic effects, hydrogen bonding,

ring current effects, etc. on the chemical shift of a certain 1H from different groups and

bonds, we can in principle estimate its chemical shift by adding all the effects together.

Identify the type of proton we have, such as aliphatic CH3, CH2, CH, olefinic CH2 or CH,

aromatic, a or b to a ketone or alcohol, belonging to an a a,b-unsaturated system, etc. They

will have a base value. Then add up the contributions from different groups attached to

carbons in the surrounding of our system to obtain the estimated chemical shift.

δH = δHbase + S contributions

13C chemical shifts of alkenes:

Dr. Suryaprakash continued his lectures to afternoon also. This time he was teaching the

extraction of NMR parameters. By the coupling of spins, we can see the splitting of

transitions. A small polarization is produced by the magnetic ,moment of the nuclei and this

is transmitted by overlapping orbitals to the other nuclei. Due to the J coupling, each peaks

in the spectrum is considered to split into various shapes like singlet, doublet, triplet etc.

There are some general rules in spin-spin coupling:

Multiplicity is given by 2nI+1 (weakly coupled spins)

The strength of the interaction, J is few Hertz to few tens of Hertz

The relative intensities within a multiplet are given by the coefficients of the binomial

expansion / Pascal triangle

The magnitude of the spin-spin coupling between the protons decrease with the

increase in the number of bonds

The splitting patterns are independent of the signs of the coupling constants

The coupling constants are independent of the magnetic field.

Dr. Suryaprakash then taught us to interpret several NMR signals. The interpretation of

signal requires knowledge of chemical shifts. Different types of NMR active nuclei lead to

same number of coupling constants. The signal of nuclei are split into subunits by he

presence of the number of protons present. These are again split according to the presence

of dueteriums used. Dr. Suryaprakash then made us predict the molecule by showing the

signals obtained for several molecules. Eg:

He then quickly concluded the classes by giving us the fundamental aspects on NMR

interpretation.

DR. HANUDATTA. S. ATREYA

A young scientist from NMR research centre, Indian Institute of Science Dr. Hanudatta.

S. Atreya continued the next spell. He made the first lecture teaching about NMR data

processing and the mathematical aspects of NMR analysis. Processing an NMR spectrum

includes mainly 5 steps:

Applying a window function (or apodization)

Zero filling the data

Fourier transformation

Phase correction

Baseline correction

Applying a weighting function is to emphasise certain portion of the FID at the cost of

other region of the spectrum. They can be used to enhance the signal to increase the

resolution. It is done by the multiplication of suitable window function with the FID. The

window function is also used for dealing with truncated airfacts. Zero filling is the addition

of zeros simply to the data we obtained. The next step is the most important Fourier

transformations using mathematical principles. Fourier transformation is a mathematical

technique to represent a signal which varies with time in a frequency scale. Performing a FT

helps us to know the frequency and the relative intensities of the different signals present.

The last steps include the phasing of spectrum and baseline correction. It can be either by

zero order phase correction or by first order phase correction. In zero order phase correction,

the same phase correction value is applied to all peaks in the spectrum while in first order

phase correction different phase correction is applied to different peaks. Dr. Atreya concluded

his 1st lecture talking about these aspects.

The next day Dr.Atreya began the classes dealing the topics of NMR spectroscopy and its

application to chemical systems. Spectroscopy involves the study of interactions of radiation

with matter. Specific wavelength of the radiation is chosen to interact with matter

depending on the property being studied. The most commonly studied nuclei are 1H, 13C,

31P,15N, 19F, 29Si etc. all of these have spins of half. The external magnetic field exerts a

torque on the spinning nucleus. This causes the nuclear spin to precess around the magnetic

field. The precessional frequency is same as the energy gap between the two spin states. At

equilibrium, the vector sum of magnetic moment in the ‘x-y’ plane is zero and a small net

moment in the +ve z direction is obtained. This net magnetic moment is termed as

magnetization.

J coupling or spin spin coupling is the interaction between nucleus separated by

covalent bonds. This gives rise to the phenomenon of multiplets of peaks. But the j coupling

strength will not change with magnetic field same like that of the chemical shift. If a given

nucleus is attached to n other nuclei with same J-coupling strength, then its peak will be

split into n + 1 number of peaks (this applies to spin-1/2 nuclei). The splitting pattern

obtained by J coupling can be understood by Pascal’s triangle. Upon each splitting, th signal

intensity is found to be reduved to half of what it was before splitting. Certain exceptions to

the n+1 rule are like the, protons that are equivalent by symmetry usually do not split one

another and the protons which are equivalent by fast rotation do not split one another. It

also, does not apply to the protons on double bonds or on benzene rings.

He managed to take an important lecture on the topic of nuclear spin relaxation which

was skipped by other professors. Relaxation is the process by which a nuclear spin system

returns to thermal equilibrium after absorption of RF energy. If RF energy is applied to the

nuclear spin system at the resonance frequency the probability of an upward transition is

equal to that of a downward transition. Because there is a greater number of nuclei in the

lower energy state, there will be more transitions from the lower energy state to the upper

state than vice versa resulting in a non equilibrium distribution of nuclear spins. Relaxation

processes, which neither emit nor absorb radiation, permit the nuclear spin system to

redistribute the population of nuclear spins. Some of these processes lead to the non-

equilibrium spin distribution (N lower – N upper) exponentially approaching the equilibrium

distribution (N lower– N upper) equil. The time constant is T1 for the exponential relaxation of the

spin system is the spin-lattice relaxation time. There are additional relaxation processes that

adiabatically redistribute any absorbed energy among the many nuclei in a particular spin

system without the spin system as a whole losing energy. Therefore, the lifetime for any

particular nucleus in the higher energy state may be decreased, but the total number of

nuclei in that state will be unchanged. This also occurs exponentially and has a time

constant T2, the spin-spin relaxation time.

For spin-1/2 nuclei, the relaxation processes occur by interaction of the nuclear spin

with magnetic fields, produced by magnetic dipoles (e.g., other nuclei, paramagnetic ions),

which are fluctuating due to random molecular motions, both rotational and translational.

The nature and the rate of the molecular motions affect the T1and T2relaxation times.

Molecular motions that occur at a rate comparable to the resonance frequency no for the

nucleus are most effective in promoting spin-lattice relaxation, i.e., yield the lowest values

for T1. T2values can be decreased even further as the molecular motion becomes slower

than no, but T1values will begin to increase.

Another parameter is the Nuclear Overhauser Effect (NOE). The nuclear Overhauser

effect or NOE is a relaxation parameter which has been used as the primary tool for

determining three-dimensional molecular structure. When two nuclei are in sufficiently close

spatial proximity, there may be an interaction between the two dipole moments. The

interaction between a nuclear dipole moment and the magnetic field generated by another was

already noted to provide a mechanism for relaxation. The nuclear dipole-dipole coupling thus

leads to the NOE as well as T1relaxation. If there is any mechanism other than from nuclear

dipole-dipole interactions leading to relaxation the NOE will be diminished – perhaps

annihilated. While it is possible to measure an NOE from a 1D NMR spectrum, usually 2D NOE

(or sometimes 3D NOE) experiments are performed. The intensities of the cross-peaks in the

spectrum depend on the distance between the interacting nuclei; it is this relationship that

provides structural information. It was Dr. Attreya who wound up the whole workshop classes

with his last class.

DR. RAGHAV G MAVINKURVE

Another class on NMR instrumentation was given by Dr. Raghav G Mavinkurve an

application engineer @ Burker India Scientific, Bangalore. He gave an introduction with the

history of NMR and then talked about the instrumentation area. NMR instrumentation mainly

consisted of four parts: magnet, console, probes &software. He demonstrated various types of

NMR instruments produced by The Bruker in various years.

The core part of NMR is the very strong electromagnet. Thin wires of NbTi or Nb3Sn

alloys are used as super conducting material as they have zero resistance at very low

temperatures. The low temperature condition can be created by immersing the superconductor

in a bath of liquid helium. We use nitrogen to minimize the evaporation of liquid helium.

Extreme care have to be given to the core magnet by various steps like always keeping magnet

ON, filling the cryogens with liquid nitrogen and helium at the appropriate time periods, never

taking any magnetic materials near the magnet to around 1.5 meters and keeping the shim

canon closed while not in use etc. those persons with iron implants and pacemakers are

advised not to go near the magnet.

Another important part is the lock. The lock channel can be understood as a ‚complete

indepenant spectrometer within the spectrometer. It has two quadrature channels which may

be either absorption or dispersion. The absorption signal is used for field homogenisation. The

signal intensity is a measure for the field homogneity: sharp signal, high lock level: broad signal,

low lock level. The dispersion signal is used for field stabilisation. If the lock phase is not

adjusted correctly, absorption and dispersion signals will be mixed. Non-pure phases will be

the result.

Console is another important part. Frequency variation is achieved by applying field of

high strength for small amount of time. Keep the magnetic field constant to get varying

frequencies. Amplifiers are part of console. It is characterised by amplification of the input

signal. In older amplifiers even the powers were set. The amplifiers can be blanked – reduces

the noise cross talk during reception of the fid. They operate over specific ranges.

Demodulation is the act of extracting the original information- bearing signal from a modulated

carrier wave. Digitizer is the analog to digital converter supplementing this.

Probes are parts which excites the NMR sample and receiving the signal. Based on the inner

coil classified as BBO or BBI. Dephasing: Gradients applied during the pulse sequence when the

magnetization lies on the transverse (xy) plane dephases the magnetization. Coherence selection:

Just prior to acquisition a final gradient applied which only refocuses the magnetization that has

gone trough the desired coherence transfer pathway. The undesired magnetization is further

dephased and not detected.

Softwares are the interface between console and the user. It can perform the processing tasks

like zero filling, fourier transform, peak picking, integration etc. It also carry out the automation of

the experiment.

REFERENCES

E. L. Hahn and D. E. Maxwell "Spin Echo Measurements of Nuclear Spin Coupling in

Molecules".

N. F. Ramsey and E. M. Purcell "Interactions between Nuclear Spins in Molecules".

Physical Review

J.W. Akitt, B.E. Mann . NMR and Chemistry. Cheltenham, UK: Stanley Thornes

NMR spectroscopy by Kalsi.