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2015.02.14. What is the medical importance of NMR? (see lecture No. 13. in this semeter!) UNIVERSITY OF PÉCS MEDICAL SCHOOL www.medschool.pte.hu NMR: Nucelar Magnetic Resonance Miklós Nyitrai, 25 February, 2015 www.medschool.pte.hu Physical bases, spins and nuclei www.medschool.pte.hu Spins and nuclei www.medschool.pte.hu Properties of nuclear spin Nucleus: proton and neutron Protons and neutrons (and also the electrons) are particles having a spin angular momentum of 1/2. If the number of protons or neutrons is add in the nucleus, then the total nuclear spin will not be zero! Two magnetic moments (spin) pointing in opposite directions zero net magnetic moment! nuclei with even Z and even N have nuclear spin I=0 (isotopes are important) If there are unpaired protons or neutrons in a nucleus, then the net nuclear spin (the intrinsic nuclear magnetic momentum) differs from zero. www.medschool.pte.hu www.medschool.pte.hu 1 2015.02.14. The split of the energy states (Zeemann) Atoms for NMR 1H, 2H, 3H, 3He, 4He, 12C, 13C, 14C, 14N, 15N 16O, 17O, 19F, 23Na, 31P, etc. α-spin state, favourable, lower energy The most frequently applied nuclei: 1H, 13C, 15N, 17O, 19F, 31P N β-spin state, unfavourable, higher energy N S S N S Signal depends on: N S Spinning nucleus with it’s magnetic field alligned with the external field. Spinning nucleus with it’s magnetic field alligned against the external field. - Magnitude of the magnetic moment The energy difference between the spin states does depend on the strength of the external magnetic field. - Concentration of the isotope www.medschool.pte.hu Orientation and reorientation on the microscopic level M=Σµi www.medschool.pte.hu Graphical representation E N N at no magnetic field there is no difference between these states β-spin state S S µ µ µ µ ∆E1 < ∆E2 µ N α-spin state S N H=0; M=0 H>0; M>0 H1 < H2 H S Remember, the energy is proportional to the frequency! m: magnetic moment of the individual atom www.medschool.pte.hu An example: CH4 www.medschool.pte.hu What is the problem with this concept? E β-spin state h x 300MHz h x 500MHz It is difficult to compare the data from two instruments with different strenghts of magnetic fields. α-spin state 0T 7,05 T 11,75 T Let’s use the chemical shift! What is this? Much simpler than it may sound… H We can probe the energy difference of the α - and β - state of the protons by irradiating them with EM radiation of just the right energy. In a magnet of 7.05 Tesla, it takes EM radiation of about 300 MHz (radio waves). So, if we bombard the molecule with 300 MHz radio waves, the protons will absorb that energy and we can measure that absorbance. In a magnet of 11.75 Tesla, it takes EM radiation of about 500 MHz (stronger magnet means greater energy difference between the α and β - state of the protons). www.medschool.pte.hu www.medschool.pte.hu 2 2015.02.14. An example: chemical shift Chemical shift or δ We need a reference sample to standardize our insturments! 1. 2. 3. 4. Measure the absorbance frequency of the standard: fr; Measure the frequency for your own sample: fs; Let’s calculate the difference between the two: ∆f = fr - fs ; Normalise the difference with the ferquency of the reference: δ = ∆f / fr ; The δ, i.e. the chemical shift will be characteristic for your sample, but will not depend on the parameters of the instrument used for the experiments! Imagine that we have a magnet where our standard absorbs at 300,000,000 Hz (300 megahertz), and our sample absorbs at 300,000,300 Hz. The difference is 300 Hz, so we take 300/300,000,000 = 1/1,000,000 and call that 1 part per million (or 1 PPM). Now lets examine the same sample in a stronger magnetic field where the reference comes at 450,000,000 Hz, or 450 megahertz. The frequency of our sample will increase proportionally, and will come at 450,000,450 Hz. The difference is now 450 Hz, but we divide by 450,000,000 (450/450,000,000 = 1/1,000,000, = 1 PPM). (We do not have to calculate all these, the NMR machine does it for us!) Why? Let’s take an example! www.medschool.pte.hu NMR spectrum www.medschool.pte.hu Applications of NMR The absorption is proportional to the concentration of the corresponding nuclei. The NMR spectrum is the energy absorbed by the system as the function of the freqeuncy (f) of the excitation energy (ΔE) or the magnetic field (H, B). Due to local effects and fields the excitation energy (and thus frequency) is different for different nuclei. Structure of organic molecules and subtances; Interactions of organic molecules; Structure of macromolecules (proteins, nucleic acids); Biological and artificial membranes; MRI: Magnetic Resonance Imaging. www.medschool.pte.hu www.medschool.pte.hu SUMMARY Thank you! www.medschool.pte.hu www.medschool.pte.hu 3