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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 Iz = +1/2 and Iz = -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 1,000,000 then Nj  999,938 N0- Nj =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 d5 term symbol is D ( -3,-2,-1,0,+1,+2,+3) ML = ± 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 MS = ±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 N2                                              Liq 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, Bo, by a fraction .                       B = Bo (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 - nREF) x106 / nREF

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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