Electron Spin Resonance, Basic principle of NMR, Application of NMR in the study of Biomolecules, NMR imaging and in vivo NMR spectromicroscopy
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1 Electron Spin Resonance, Basic principle of NMR, Application of NMR in the study of Biomolecules, NMR imaging and in vivo NMR spectromicroscopy Mitesh Shrestha
2 Electron Spin Resonance Electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy is a method for studying materials with unpaired electrons. Particularly useful for studying metal complexes or organic radicals.
3 What is EPR? Electron Paramagnetic Resonance also known as Electron Spin Resonance is a spectroscopic technique which detect species that have unpaired electrons. A surprisingly large number of materials have unpaired electrons. (examples: free radicals and many transition metal ions) EPR alone yields inconvertible evidence of free radicals and has the unique power of identifying the paramagnetic species. Emblem of the Society of Free Radical Research
4 EPR Instruments The EPR Spectrometer consists of many different components. Microwave Bridge: Electromagnetic Radiation Source Microwave Cavity: Amplifies weak signals from the sample Magnet: Splits the electronic spin energy levels Console: Contains signal processing and control electronics and a units for acquiring a spectrum Computer: Used to analyze data as well as coordinating all the units for acquiring a spectrum.
5 How does EPR Work? Like a proton, an electron has a spin, which gives it a magnetic property known as a magnetic moment. When an external magnetic field is supplied, the paramagnetic electrons can either orient in a direction parallel or antiparallel to the direction of the magnetic field. This creates two distinct energy levels for the unpaired electrons and measurements are taken as they are driven between the two levels.
6 EPR Spectroscopy EPR spectroscopy is the measurement and interpretation of the energy differences between the atomic or molecular states. These measurements are obtained because the relationship between the energy differences and the absorption of electro-magnetic radiation. To acquire a spectrum, the frequency of the electromagnetic radiation is changed and the amount of radiation which passes through the sample with a detector is measured to observe the spectroscopic absorptions
7 Electron Spin Resonance 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.
8 ESR Spectroscopy Uses microwave radiation on species that contain unpaired electrons placed ina magnetic fieled 1.Free radicals 2.Odd electron molecules 3.Transition-metal complexes 4.Lanthanide ions 5.Triplet-state molecules
9 Application For the detection and identification of free radicals and paramagnetic centers such as F-centers. Sensitive and specific method for studying both radicals formed in chemical reactions and the reactions themselves. For example, when ice (solid H2O) is decomposed by exposure to high-energy radiation, radicals such as H, OH, and HO 2 are produced. Such radicals can be identified and studied by EPR. Organic and inorganic radicals can be detected in electrochemical systems and in materials exposed to UV light. In many cases, the reactions to make the radicals and the subsequent reactions of the radicals are of interest, while in other cases EPR is used to provide information on a radical's geometry and the orbital of the unpaired electron. EPR/ESR spectroscopy is also used in geology and archaeology as a dating tool. It can be applied to a wide range of materials such as carbonates, sulfates, phosphates, silica or other silicates.
10 Application Electron spin resonance has been used as an investigative tool for the study of radicals formed in solid materials, since the radicals typically produce an unpaired spin on the molecule from which an electron is removed. Particularly fruitful has been the study of the ESR spectra of radicals produced as radiation damage from ionizing radiation. Study of the radicals produced by such radiation gives information about the locations and mechanisms of radiation damage.
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12 ESR is one of the most powerful tools in lipid research Phase state of lipids Interaction of lipid with proteins, formation of lipoproteids, boundary lipid etc. Domains in model and biological membranes Diffusion studies in the membrane phase Polarity profiles in membranes Membrane permeation profiles for oxygen and paramagnetic ions
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14 Nuclear Magnetic Resonance (NMR) spectroscopy
15 Nuclear Magnetic Resonance (NMR) spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical chemistry technique used in quality control and research for determining the content and purity of a sample as well as its molecular structure. For example, NMR can quantitatively analyze mixtures containing known compounds. For unknown compounds, NMR can either be used to match against spectral libraries or to infer the basic structure directly. Once the basic structure is known, NMR can be used to determine molecular conformation in solution as well as studying physical properties at the molecular level such as conformational exchange, phase changes, solubility, and diffusion. In order to achieve the desired results, a variety of NMR techniques are available.
16 Introduction to NMR Spectroscopy Nuclear magnetic resonance spectroscopy is a powerful analytical technique used to characterize organic molecules by identifying carbon-hydrogen frameworks within molecules. Two common types of NMR spectroscopy are used to characterize organic structure: 1 H NMR is used to determine the type and number of H atoms in a molecule; 13 C NMR is used to determine the type of carbon atoms in the molecule. The source of energy in NMR is radio waves which have long wavelengths, and thus low energy and frequency. When low-energy radio waves interact with a molecule, they can change the nuclear spins of some elements, including 1 H and 13 C.
17 Introduction to NMR Spectroscopy When a charged particle such as a proton spins on its axis, it creates a magnetic field. Thus, the nucleus can be considered to be a tiny bar magnet. Normally, these tiny bar magnets are randomly oriented in space. However, in the presence of a magnetic field B0, they are oriented with or against this applied field. More nuclei are oriented with the applied field because this arrangement is lower in energy. The energy difference between these two states is very small (<0.1 cal).
18 Nuclear Magnetic Resonance Spectroscopy Introduction to NMR Spectroscopy In a magnetic field, there are now two energy states for a proton: a lower energy state with the nucleus aligned in the same direction as B0, and a higher energy state in which the nucleus aligned against B0. When an external energy source (hn) that matches the energy difference (DE) between these two states is applied, energy is absorbed, causing the nucleus to spin flip from one orientation to another. The energy difference between these two nuclear spin states corresponds to the low frequency RF region of the electromagnetic spectrum.
19 Introduction to NMR Spectroscopy Thus, two variables characterize NMR: an applied magnetic field B0, the strength of which is measured in tesla (T), and the frequency n of radiation used for resonance, measured in hertz (Hz), or megahertz (MHz) (1 MHz = 10 6 Hz).
20 Nuclear Magnetic Resonance Spectroscopy Introduction to NMR Spectroscopy The frequency needed for resonance and the applied magnetic field strength are proportionally related: NMR spectrometers are referred to as 300 MHz instruments, 500 MHz instruments, and so forth, depending on the frequency of the RF radiation used for resonance. These spectrometers use very powerful magnets to create a small but measurable energy difference between two possible spin states.
21 Basic NMR Spectrometer
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23 Nuclear Magnetic Resonance Spectroscopy Introduction to NMR Spectroscopy
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26 Nuclear Magnetic Resonance Spectroscopy Introduction to NMR Spectroscopy Protons in different environments absorb at slightly different frequencies, so they are distinguishable by NMR. The frequency at which a particular proton absorbs is determined by its electronic environment. The size of the magnetic field generated by the electrons around a proton determines where it absorbs. Modern NMR spectrometers use a constant magnetic field strength B 0, and then a narrow range of frequencies is applied to achieve the resonance of all protons. Only nuclei that contain odd mass numbers (such as 1 H, 13 C, 19 F and 31 P) or odd atomic numbers (such as 2 H and 14 N) give rise to NMR signals.
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28 Nuclear Magnetic Resonance (NMR) spectroscopy The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged. If an external magnetic field is applied, an energy transfer is possible between the base energy to a higher energy level (generally a single energy gap). The energy transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned.
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34 Information in a NMR Spectra 1) Energy E = hu h is Planck constant u is NMR resonance frequency g-rays x-rays UV VIS IR m-wave radio wavelength (cm) Observable Name Quantitative Information Peak position Chemical shifts (d) d(ppm) = u obs u ref /u ref (Hz) chemical (electronic) environment of nucleus Peak Splitting Coupling Constant (J) Hz peak separation neighboring nuclei (intensity ratios) (torsion angles) Peak Intensity Integral unitless (ratio) nuclear count (ratio) relative height of integral curve T 1 dependent Peak Shape Line width Du = 1/pT 2 molecular motion peak half-height chemical exchange uncertainty principal uncertainty in energy
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38 Hydrogen and Carbon Chemical Shifts =>
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46 The N + 1 Rule If a signal is split by N equivalent protons, it is split into N + 1 peaks. =>
47 Interpreting 13 C NMR The number of different signals indicates the number of different kinds of carbon. The location (chemical shift) indicates the type of functional group. The peak area indicates the numbers of carbons (if integrated). The splitting pattern of off-resonance decoupled spectrum indicates the number of protons attached to the carbon. =>
48 NMR Sensitivity NMR signal depends on: signal (s) g 4 B o2 NB 1 g(u)/t 1) Number of Nuclei (N) (limited to field homogeneity and filling factor) 2) Gyromagnetic ratio (in practice g 3 ) 3) Inversely to temperature (T) 4) External magnetic field (B 2/3 o, in practice, homogeneity) 5) B 2 1 exciting field strength N a / N b = e DE / kt DE = g h B o / 2p Increase energy gap -> Increase population difference -> Increase NMR signal DE B o g g - Intrinsic property of nucleus can not be changed. (g H /g C ) 3 for 13 C is 64x (g H /g N ) 3 for 15 N is 1000x 1 H is ~ 64x as sensitive as 13 C and 1000x as sensitive as 15 N! Consider that the natural abundance of 13 C is 1.1% and 15 N is 0.37% relative sensitivity increases to ~6,400x and ~2.7x10 5 x!!
49 Nuclear Magnetic Resonance Spectroscopy 1 H NMR Structure Determination 49
50 Nuclear Magnetic Resonance Spectroscopy 1 H NMR Structure Determination 50
51 Nuclear Magnetic Resonance Spectroscopy 1 H NMR Structure Determination 51
52 Nuclear Magnetic Resonance Spectroscopy 1 H NMR Structure Determination 52
53 MRI Magnetic resonance imaging, noninvasive Nuclear is omitted because of public s fear that it would be radioactive. Only protons in one plane can be in resonance at one time. Computer puts together slices to get 3D. Tumors readily detected.
54 Magnetic Resonance Imaging (MRI) 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, MRimaging 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.
55 Videos Important
56 Thanks a lot everyone For having such an amount of patience for my flaws And just pray that I too have got the same for yours.. It was fun when it lasted.. And now I just have this thing to say.. You know my name [Hint: First Slide].. Its not going to change.. Hence, if you have any problem.. Just send me message or and I ll try my best to help you.. Best of luck [you are going to need it] for the exam..
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