* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Multinuclear NMR Notes
Survey
Document related concepts
Lorentz force wikipedia , lookup
Neutron magnetic moment wikipedia , lookup
Electric charge wikipedia , lookup
Coherence (physics) wikipedia , lookup
Aharonov–Bohm effect wikipedia , lookup
Magnetic monopole wikipedia , lookup
Superconductivity wikipedia , lookup
Electrostatics wikipedia , lookup
Time in physics wikipedia , lookup
Thought experiment wikipedia , lookup
Circular dichroism wikipedia , lookup
Nuclear binding energy wikipedia , lookup
Nuclear drip line wikipedia , lookup
Nuclear structure wikipedia , lookup
Transcript
1 Multinuclear NMR Notes 4/1/2010 NOTE: We will be discussing a paper on sodium MR in cartilage from the MRI lab in the final section of class. This reference is as follows. Shapiro et. al. “Sodium Visibility and Quantation in Intact Bovine Articular Cartilage Using High Field 23NA MRI and MRS”. Journal of Magnetic Resonance. 142, 24-31 (2000) I. Properties of NMR-Active Nuclei 1H and 13C are the most commonly used nuclei in NMR, but almost all elements have stable NMR-active nuclei. Not all nuclei, however, are equally easy to observe. There are several factors that influence signal strength: A) Isotopic abundance—if the abundance of an isotope is too low, it won’t give a signal. You may need to isotopically enrich the sample. B) Chemical shift range—if the chemical shift range is too large, that means that the nucleus is very sensitive to its environment; relatively minor inhomogeneities in the sample (such as a temperature gradient) can create significant line broadening. The chemical shift range of 1H is about 10 ppm (low extreme), and that of Pb is about 20000 ppm (high extreme). C) T2 length—since line width equals 1/(πT2), a short T2 will give a broad line width, and may not be very useful. D) Receptivity—also called sensitivity, the relative signal strength of a nucleus as compared to 1H or 13C under the same conditions. Receptivity of nuclide X (RX) is given by RX = A∙|γ3|∙I(I+1) Receptivity has a cubic dependence on γ because γ is proportional to |M0|, ΔE, and ω0, as shown in the equations below. 1) M= γ∙P, where M is the total magnetization of the sample and P is the total spin angular momentum. 2) ΔE=h γ B0, where h is Planck’s constant, B0 is the magnitude of the applied magnetic field, and ΔE is the energy difference between spin states. 2 3) ω0=-γ B0, where ω0 is the Larmor frequency. Equations 1) and 2) above influence the magnitude of the magnetization that is detected, and equation 3) is relevant because the induced current in the coil is proportional to the rate of change of the magnetic moment, so a faster Larmor frequency gives a stronger signal. Below is a table containing relative receptivities. (DP is sensitivity relative to 1H, and DC is sensitivity relative to 13C.) This table is from http://www.stanford.edu/group/chem-NMR/chem235_lecture_notes/lecture_1.pdf 3 E) 4 Nuclear Electric Quadrupole Moment—a nuclear quadrupole broadens the line width. Not all nuclei are spheres. Some have prolate or oblate shapes. Only non-spherical nuclei have quadrupoles. The charge distribution of a nucleus can be described by a superposition of electric multipoles. These multipoles have the same shapes as s, p, d, etc. atomic orbitals. C(r) represents the charge distribution of the nucleus. C(r) = C(0)(r) + (total charge) C(1)(r) (dipole) + C(2)(r) (quadrupole) Note: • No nuclide has an electric dipole moment • The spherical C(0)(r) term has no direct significance in NMR • C(n)(r) = 0 for n > 2I All spin-1/2 nuclei . . . – behave exactly like point charges located at the nuclear center (as far as electrical effects) – have a spherical charge distribution – have no nuclear electric dipole, quadrupole, etc. – lack electric energy terms that depend on orientation Nuclides with I>1/2 . . . – are termed “quadrupolar” – have non-spherical charge distribution – have an electric energy that is dependent on orientation in an electric field gradient The degree of broadening due to the quadrupole moment can be measured by the linewidth factor, l, according to l=Q2(2I+3) I2(2I-1) A nuclide can assume 2I+1 different orientations in a magnetic field. For quadrupolar interactions, the energy separation between states is given by ΔE = 2πe2qQ(3cos2θ-1)/(h∙4), where e=elementary charge, q=electric field gradient, Q=quadrupole moment, and θ=angle between q and B0. 5 II) Discussion of several multi-quantum NMR experiments. This section offers a quick overview of several 2d and 3d NMR experiments for determination of molecular structure. For each experiment we will discuss the pulse sequences and resultant data. Homonuclear (1H, multi-dimensional) and Heteronuclear (1H plus one or more additional nuclei) experiments will be addressed. We emphasize solution protein NMR, but in principle these techniques can be applied to other organic as molecules as well. A) HSQC: Heteronuclear Single Quantum Coherence. This 2d experiment measures coherence between protons and a single directly-bonded heteronucleus. Typically, the amide nitrogen and hydrogen in the protein backbone will be measured. This is sometimes referred to as a “protein fingerprint”. The results allow one to estimate which residues as present, and can be used as a “sanity check” to match a protein sequence obtained through biochemical methods. The HSQC does not provide information on residue order or three-dimensional structure. B) COSY: Correlation Spectroscopy. Ravi covered this method in class on 3/30. COSY is a 2-d homonuclear experiment to measure J-coupling between protons separated by up to 3 bonds. The two dimensions (t1 and t2) of the resultant spectrum refer to the “evolution time” between two pulses, and the “detection time” after the second pulse. The experiment is carried out as a series of 1d FID measurement, each with a slightly different and carefully controlled value for the evolution time. COSY can be used to estimate the phi angles between amide N and alpha C in the protein backbone. C) TOCSY: Totally Correlated Spectroscopy. This 2d homonuclear experiment can be viewed as a “longer-range COSY.” It incorporates a spin-lock pulse to extend coherence beyond 3 bonds. This is useful in molecules containing carbon rings such as sugars. 6 D) NOESY: Nuclear Overhauser Effect Spectroscopy. Another 2-d homonuclear experiment. This adds a third pulse to the COSY pulse sequence, allowing nuclear spins to cohere through space rather than through bonds. The throughspace coupling falls off with the 6th power of distance and generally extends to about 5 nm. NOESY is useful on examining 3d structure, as it allows one to observe protons that are spatially close but perhaps widely separated in sequence. Such information provides insight into protein tertiary structure. E) NOESY – HSQC: A 3d experiment combining both thru-space proton coupling and heteronuclear J-coupling. Similar 3d experiments can by devised by combining other 2d homonuclear and 2d heteronuclear methods to obtain a 3d spectrum. F) Triple Resonance Experiments. A variety of experiments can be carried out to examine coherence between 1H, 13C, and 15N nuclei. Using 3 frequencies rather than 2 makes the spectrum simpler to interpret by reducing “overlap” in the data. The nomenclature for these experiments describes the pattern of spin coherence that they measure. Consider the example CBCA(CO)NH. Spin is transferred as follows: C on side-chain C in backbone carboxyl C amide N (across peptide bond) amide H. II) 23 Na Imaging in Cartilage Shapiro et. al. “Sodium Visibility and Quantation in Intact Bovine Articular Cartilage Using High Field 23NA MRI and MRS”. Journal of Magnetic Resonance. 142, 24-31 (2000) This study examines a novel imaging technique based on sodium magnetic resonance. It is not a multi-quantum technique per se, but it does illustrate an interesting application taking advantage of the properties of a “different” nucleus. 7 Osteoarthritis is a disease characterized by the degradation of cartilage, specifically loss of proteoglycan (PG) in the extracellular matrix. PG contains a large number of negative charges which are balanced though electrostatic interactions with intercellular sodium. Osmotic forces then lead water to “follow” the Na+ into the tissue. This study demonstrates an MRI method to provide a map of sodium concentration in tissue. A decline in sodium density may correlate to proteoglycan loss, a precursor to osteoarthritis. A major question is the “visibility” of sodium in cartilage – for a given concentration of sodium, what is the intensity of the MR signal in tissue vs. simple solution? Association with biomolecules may enhance T2 decay, decreasing visibility. For other tissues, sodium visibility has been shown to be significantly less than 100% • • • Muscle: ~40% Cartilage: ~100% (but method was questionable) Some others: ~40-90% Sodium NMR was run on solid cartilage plugs. The plugs were then liquefied in acid and the experiment repeated. The results implied the same sodium quantities in each, illustrating that sodium visibility does not decrease in cartilage vs. solution. Phantoms were constructed consisting of agarose (to simulate proteoglycan) and saline. These were used to calibrate MRI to obtain sodium concentration at various location in bovine patellae, as shown below: