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NUCLEAR MAGNATIC RESONANCE SPECTROSCOPY INTRODUCTION BASICS PRINCIPLE INSTRUMENTATION SHEILDING AND DESHIELDING &APPLICATIONS INTRODUCTION Nuclear magnetic resonance (NMR) is a physical phenomenon based upon the quantum mechanical magnetic properties of an atom's nucleus. NMR also commonly refers to a family of scientific methods that exploit nuclear magnetic resonance to study molecules ("NMR spectroscopy"). The method of NMR was first developed by E.M. Purcell and Felix bloch(1946) Major application of NMR spectroscopy lies in the area of synthetic organic chemistry, inorganic chemistry, bio-organic chemistry, bioinorganic chemistry, NMR Historic Review NMR Historic Review 1924 Pauli proposed the presence of nuclear magnetic moment to explain the hyperfine structure in atomic spectral lines. 1930 Nuclear magnetic moment was detected using refined Stern-Gerlach experiment by Estermann. 1939 Rabi et al. First detected unclear magnetic resonance phenomenon by applying r.f. energy to a beam of hydrogen molecules in the Stern-Gerach set up and observed measurable deflection of the beam. 1946 Purcell et al. at Harvard reported nuclear resonance absorption in paraffin wax. Bloch et al. at Stanford found nuclear resonance in liquid water. 1949 Chemical shift phenomenon was observed. 1952 Nobel prize in Physics was awarded to Purcell and Bloch. 1966 Ernst and Anderson first introduce the Fourier Transform technique into NMR. NMR. Late in the 1960s: Solid State NMR was revived due to the effort of Waugh. and associates at MIT. Biological application become possible due to the introduction superconducting magnets. NMR imaging was demonstrated. 1970 2D NMR was introduced. 1980s Macromolecular structure determination in solution by NMR was achieved. 1991 Nobel prize in Chemistry was awarded to Richard Ernst. 1990s Continuing development of heteronuclear multi-dimensional NMR permit the determination of protein structure up to 50 KDa. MRI become a major radiological tool in medical diagnostic. 2002 NMR Applications Nobel prize in Chemistry was awarded to Kurt Wuthrich (One of themost, if not the most, important analytical spectroscopic tool.) 1. Biomedical applications: Edward M. Purcell 1912-1997 Felix Bloch 1905-1983 Richard R. Ernst 1933- Kurt Wuthrich 1938- CW NMR 40MHz 1960 800 MHz NMR Spectroscopy Where is it? 1nm (the wave) Frequency (the transition) (spectrometer) 102 10 X-ray 103 UV/VIS electronic X-ray 104 Infrared Vibration UV/VIS Infrared/Raman Fluorescence 105 106 Microwave Rotation 107 Radio Nuclear NMR Before using NMR What are N, M, and R ? Properties of the Nucleus Nuclear spin Nuclear magnetic moments The Nucleus in a Magnetic Field Precession and the Larmor frequency Nuclear Zeeman effect & Boltzmann distribution When the Nucleus Meet the right Magnet and radio wave Nuclear Magnetic Resonance Nuclear magnetic moments Magnetic moment is another important parameter for a nuclei = I (h/2) I: spin number; h: Plank constant; : gyromagnetic ratio (property of a nuclei) 1H: I=1/2 , = 267.512 *106 rad T-1S-1 13C: I=1/2 , = 67.264*106 15N: I=1/2 , = 27.107*106 PRINCIPLE • Subatomic particles (electrons, protons and neutrons) can be imagined as spinning on their axes. • In many atoms (such as 12C) these spins are paired against each other, such that the nucleus of the atom has no overall spin. • However, in some atoms (such as 1H and 13C) the nucleus does possess an overall spin. The rules for determining the net spin of a nucleus are as follows; 1. If the number of neutrons and the number of protons are both even, then the nucleus has NO spin. 2 . If the number of neutrons plus the number of protons is odd, then the nucleus has a half-integer spin (i.e. 1/2, 3/2, 5/2) 3.If the number of neutrons and the number of protons are both odd, then the nucleus has an integer spin (i.e. 1, 2, 3) Natural % Abundanc e Isotope Spin (I) Magnetic Moment (μ)* Magnetogyri c Ratio (γ)† 1H 99.9844 1/2 2.7927 26.753 2H 0.0156 1 0.8574 4,107 11B 81.17 3/2 2.6880 -- 13C 1.108 1/2 0.7022 6,728 17O 0.037 5/2 -1.8930 -3,628 19F 100.0 1/2 2.6273 25,179 29Si 4.700 1/2 -0.5555 -5,319 31P 100.0 1/2 1.1305 10,840 1. A spinning charge generates a magnetic field.The resulting spinmagnet has a magnetic moment (μ) proportional to the spin. 2. In the presence of an external magnetic field (B0), two spin states exist, +1/2 and -1/2. 3.The magnetic moment of the lower energy +1/2 state is aligned with the external field, but that of the higher energy -1/2 spin state is opposed to the external field. Note that the arrow representing the external field points North. In an Applied Magnetic Field •Nuclei with 2 allowed spin states can align either with or against the field, with slight excess of nuclei aligned with the field •The nuclei precess about an axis parallel to the applied magnetic field, with a frequency called the Larmor Frequency (w) Larmor Frequency is Proportional to the Applied Magnetic Field Slow precession in small magnetic field Faster precession in larger magnetic field Nuclear Zeeman effect • Zeeman effect: when an atom is placed in an external magnetic field, the energy levels of the atom are split into several states. • The energy of a give spin sate (Ei) is directly proportional to the value of mI and the magnetic field strength B0 Spin State Energy EI=- . B0 =-mIB0 r(h/2p) •For a nucleus with I=1/2, the energy difference between two states is ΔE=E-1/2-E+1/2 = B0 r(h/2p) m=1/2 m=-1/2 The Zeeman splitting is proportional to the strength of the magnetic field Boltzmann distribution Quantum mechanics tells us that, for net absorption of radiation to occur, there must be more particles in the lower-energy state than in the higher one. If no net absorption is possible, a condition called saturation. When it’s saturated, Boltzmann distribution comes to rescue: Pm=-1/2 / Pm=+1/2 = e -DE/kT where P is the fraction of the particle population in each state, T is the absolute temperature, k is Boltzmann constant 1.381*10-28 JK-1 Anything that increases the population difference will give rise to a more intense NMR signal. Nuclear Magnetic Resonance Spectrometer How to generate signals? B0: magnet B1: applied small energy Rf Energy Can Be Absorbed •Precessing nuclei generates an oscillating electric field of the same frequency •Rf energy with the same frequency as the Larmor frequency can be applied to the system and absorbed by the nuclei The Nucleus in a Magnetic Field Precession and the Larmor frequency • The magnetic moment of a spinning nucleus processes with a characteristic angular frequency called the Larmor frequency w, which is a function of r and B0 Remember = I (h/2) ? Angular momentum dJ/dt= x B0 Larmor frequency w=rB0 Linear precession frequency v=w/2p= rB0/2p J When the Nucleus Meet the Magnet Nuclear Magnetic Resonance •For a particle to absorb a photon of electromagnetic radiation, the particle must first be in some sort of uniform periodic motion • If the particle “uniformly periodic moves” (i.e. precession) at precession, and absorb erengy. The energy is E=hvprecession v •For I=1/2 nuclei in B0 field, the energy gap between two spin states: DE=rhB0/2p DE =hvphoton • The radiation frequency must exactly match the precession frequency Ephoton=hvprecession=hvphoton=DE=rhB0/2p This is the so called “ Nuclear Magnetic RESONANCE”!!!!!!!!! Magnet B0 and irradiation energy B1 B0 ( the magnet of machine) (1) Provide energy for the nuclei to spin Ei=-miB0 (rh/2p) Larmor frequency w=rB0 (2) Induce energy level separation (Boltzmann distribution) The stronger the magnetic field B0, the greater separation between different nuclei in the spectra Dv =v1-v2=(r1-r2)B0/2p (3) The nuclei in both spin states are randomly oriented around the z axis. M z=M, Mxy=0 ( where M is the net nuclear magnetization) What happen before irradiation • Before irradiation, the nuclei in both spin states are processing with characteristic frequency, but they are completely out of phase, i.e., randomly oriented around the z axis. The net nuclear magnetization M is aligned statically along the z axis (M=Mz, Mxy=0) What happen during irradiation When irradiation begins, all of the individual nuclear magnetic moments become phase coherent, and this phase coherence forces the net magnetization vector M to process around the z axis. As such, M has a component in the x, y plan, Mxy=Msina. a is the tip angle which is determined by the power and duration of the electromagnetic irradiation. z Mo a x x B1 wo y y a deg pulse Mxy 90 deg pulse B1(the irradiation magnet, current induced) (1) Induce energy for nuclei to absorb, but still spin at w or vprecession Ephoton=hvphoton=DE=rhB0/2p=hvprecession And now, the spin jump to the higher energy ( from m= –1/2 m=1/2m= – 1/2) m= 1/2 (2) All of the individual nuclear magnetic moments become phase coherent, and the net M process around the z axis at a angel M z=Mcosa Mxy=Msina. What happen after irradiation ceases •After irradiation ceases, not only do the population of the states revert to a Boltzmann distribution, but also the individual nuclear magnetic moments begin to lose their phase coherence and return to a random arrangement around the z axis. (NMR spectroscopy record this process!!) •This process is called “relaxation process” •There are two types of relaxation process : T1(spin-lattice relaxation) & T2(spin-spin relaxation) • Relaxation processes • How do nuclei in the higher energy state return to the lower state? • Emission of radiation is insignificant because the probability of reemission of photons varies with the cube of the frequency. At radio frequencies, re-emission is negligible. • Ideally, the NMR spectroscopist would like relaxation rates to be fast - but not too fast. • If the relaxation rate is fast, then saturation is reduced. If the relaxation rate is too fast, line-broadening in the resultant NMR spectrum is observed. • There are two major relaxation processes; • Spin - lattice (longitudinal) relaxation • Spin - spin (transverse) relaxation T1 (the spin lattice relaxation) • How long after immersion in a external field does it take for a collection of nuclei to reach Boltzmann distribution is controlled by T1, the spin lattice relaxation time. (major Boltzmann distribution effect) •Lost of energy in system to surrounding (lattice) as heat ( release extra energy) •It’s a time dependence exponential decay process of Mz components dMz/dt=-(Mz-Mz,eq)/T1 T2 (the spin –spin relaxation) •This process for nuclei begin to lose their phase coherence and return to a random arrangement around the z axis is called spin-spin relaxation. •The decay of Mxy is at a rate controlled by the spin-spin relaxation time T2. dMx/dt=-Mx/T2 dMy/dt=-My/T2 dephasing NMR Parameters 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, = (n - nREF) x106 / nREF • In NMR spectroscopy, this standard is often tetramethylsilane, Si(CH3)4, abbreviated TMS, or 2,2-dimethyl-2-silapentane-5-sulfonate, DSS, in biomolecular NMR. • The good thing is that since it is a relative scale, the d for a sample in a 100 MHz magnet (2.35 T) is the same as that obtained in a 600 MHz magnet (14.1 T). Alcohols, protons a Deshielded (low field) Acids Aldehydes Aromatics Amides to ketones Olefins Aliphatic Shielded (up field) ppm 15 10 7 5 2 0 TMS The NMR scale (, ppm) • We can use the frequency scale as it is. The problem is that since Bloc is a lot smaller than Bo, the range is very small (hundreds of Hz) and the absolute value is very big (MHz). • We use a relative scale, and refer all signals in the spectrum to the signal of a particular compound. w - wref = ppm (parts per million) wref The good thing is that since it is a relative scale, the in a 100 MHz magnet (2.35 T) is the same as that obtained for the same sample in a 600 MHz magnet (14.1 T). • CH3 H 3C Si CH3 CH3 Tetramethyl silane (TMS) is used as reference because it is soluble in most organic solvents, inert, volatile, and has 12 equivalent 1Hs and 4 equivalent 13Cs: Other references can be used, such as the residual solvent peak, dioxane for 13C, etc. What reference we use is not critical, because the instrument (software/hardware) is calibrated internaly. Don’t use them if you don’t need to... HO-CH2-CH3 w0=rBeffect low field wo high field • Notice that the intensity of peak is proportional to the number of H J-coupling •Nuclei which are close to one another could cause an influence on each other's effective magnetic field. If the distance between non-equivalent nuclei is less than or equal to three bond lengths, this effect is observable. This is called spin-spin coupling or J coupling. 1 H 13 1 1 H H three-bond C one-bond •Each spin now seems to has two energy ‘sub-levels’ depending on the state of the spin it is coupled to: J (Hz) ab I S bb S I aa ba I S The magnitude of the separation is called coupling constant (J) and has units of Hz. •N neighboring spins: split into N + 1 lines Single spin: One neighboring spins: - CH – CH - Two neighboring spins: - CH2 – CH - • From coupling constant (J) information, dihedral angles can be derived ( Karplus equation) 3 J NHa 6.4 cos 2 ( 60) 1.4 cos( 60) 1.9 3 J ab 1 9.5 cos 2 ( 1 120) 1.6 cos( 1 120) 1.8 3 J ab 2 9.5 cos 1 1.6 cos 1 1.8 χ2 χ1 2 N Cγ Cβ Cα ψΨ N ω C’ Nuclear Over Hauser Effect (NOE) •The NOE is one of the ways in which the system (a certain spin) can release energy. Therefore, it is profoundly related to relaxation processes. In particular, the NOE is related to exchange of energy between two spins that are not scalarly coupled (JIS = 0), but have dipolar coupling. • The NOE is evidenced by enhancement of certain signals in the spectrum when the equilibrium (or populations) of other nearby are altered. For a two spin system, the energy diagram is as following: bb W1I ab W2I W0IS W1S W1S ba S W1I aa •W represents a transition probability, or the rate at which certain transition can take place. For example, the system in equilibrium, there would be W1I and W1S transitions, which represents single quantum transitions. INSTRUMENTATION 1. MAGNET Permanent magnets Conventional electromagnets and Super conducting magnets 2. SAMPLE PROBE 3. FIELD SWEEP GENARETOR 4. THE RADIO FREQUENCY SOURCE 5. THE SIGNAL DETECTOR& RECORDER SYSTEM The spectrometer Preparation for NMR Experiment 1. Sample preparation Which buffer to choose? Isotopic labeling? Best temperature? Sample Position ? N 2. S What’s the nucleus or prohead? A nucleus with an even mass A and even charge Z nuclear spin I is zero Example: 12C, 16O, 32S No NMR signal A nucleus with an even mass A and odd charge Z integer value I Example: 2H, 10B, 14N NMR detectable A nucleus with odd mass A I=n/2, where n is an odd integer Example: 1H, 13C, 15N, 31P NMR detectable 3. The best condition for NMR Spectrometer? Wobble : Tune & Match & Shimming Tune Match RCVR 0% Absorption 100% 4. Frequency What’s the goal? Which type of experiment you need? Different experiments will result in different useful information 5. NMR Data Processing The FID (free induction decay) is then Fourier transform to frequency domain to obtain vpression ( chemical shift) for each different nuclei. Time (sec) frequency (Hz) roperties of Some Deuterated NMR Solvents Solvent B.P. °C Residual 1H signal (δ) Residual 13C signal (δ) acetone-d6 55.5 2.05 ppm 206 & 29.8 ppm acetonitrile-d3 80.7 1.95 ppm 118 & 1.3 ppm benzene-d6 79.1 7.16 ppm 128 ppm chloroform-d 60.9 7.27 ppm 26.4 ppm cyclohexane-d12 78.0 1.38 ppm 26.4 ppm dichloromethane-d2 40.0 5.32 ppm 53.8 ppm dimethylsulfoxide-d6 190 2.50 ppm 39.5 ppm nitromethane-d3 100 4.33 ppm 62.8 ppm pyridine-d5 114 7.19, 7.55 & 8.71 ppm 150, 135.5 & 123.5 ppm tetrahydrofuran-d8 65.0 1.73 & 3.58 ppm 67.4 & 25.2 ppm Limitations of nmr spectroscopy 1.Its lack of sensitivity. fairly large numbers are requried.minimum sample size is about0.1ml having minimum concentrations of about on1% 2.Limited number of nuclei which may be usefully studied with this technique. 3.Inmost of the cases ,the technique is limited to liquid samples or to a liquid capable of solutions in a suitable solvents or of melting at a temperature below 260oc 4.In some compounds two different types of hydrogen atoms resonance at similar resonance frequencies .this results in an overlap of spectra .hence the interpretation of spectra becomes difficult. APPLICATIONS 1.determination of optical purity 2.study of molecular interactions 3.quantative analysis: assay components, surfactant chain length Determination, hydrogen analysis, iodine value, moisture analysis 4.elemental analysis 5.Multicomponentmixture analysis 6.magnetic resonance imaging 7. NMR has also been used in various special fields that includes industrial quality control, biology, engineering and medicine 8.Structure elucidation • • other applications Molecular conformation in solution Quantitative analysis of mixtures containing known compounds Determining the content and purity of a sample Through space connectivity (over Hauser effect) Chemical dynamics (Line shapes, relaxation phenomena) Solid State NMR is widely popular for the characterization of polymers, rubbers, ceramics, and molecular sieves. Thank you