NMR SPECTROSCOPY WORKSHOP

(24- 27 January 2013)

JEESU GEORGE MS- 2012/05

By NMR Research Centre, IISc , Bangalore.

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 Paulis 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 Wtrich from the fields of chemistry (1991, 2002); Paul C. Lauterbur and Sir Peter Mansfield (2003). He also didnt 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 = I h /2. By applying a magnetic field to an atom, the electrons

shift either to the ground state or excited state. There is an energy difference E 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 = Bo / 2 . 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 included,

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

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. T 1 is the variable delay time and T2 is the normal acquisition time. The signal along the T 2 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 T 2. 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 E 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 = is denoted and is sometimes described as spin up. The state with m = - is denoted and is sometimes described as spin down. There is variations in the case of energy levels

for homo and hetero- nuclear species. The and states have same energy in homonuclear 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 Lambs formula, shielding constant

By breaking the equation into different terms, we can understand most of the effects. By knowing the (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. H = Hbase + S contributions
13

C 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,
31

P,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 Pascals 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 nonequilibrium spin distribution (N
lower

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 T 1and 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.