Nmr Spectroscopy In B Iology


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Nmr Spectroscopy In B Iology

  2. 2. CONTENTS <ul><li>Introduction </li></ul><ul><li>Basic principles in NMR spectroscopy </li></ul><ul><li>Instrumentation </li></ul><ul><li>Interpretation of NMR spectra </li></ul><ul><li>Applications in the field of biology </li></ul>
  3. 3. Introduction
  4. 4. introduction <ul><li>History </li></ul><ul><ul><ul><li>First described and measured by Isidor Rabi in 1938. </li></ul></ul></ul><ul><ul><ul><li>In 1946, Felix Bloch & Edward Mills Purcell refined the technique for use on liquids & solids for which they shared the Nobel Prize in physics in 1952. </li></ul></ul></ul><ul><ul><ul><li>The observation of resonance frequencies of a molecule allows a user to discover structural information about a molecule. </li></ul></ul></ul>Contd…
  5. 5. Basic Principles in NMR Spectroscopy
  6. 6. basic principle in NMR spectroscopy <ul><li>Magnetic phenomena </li></ul><ul><ul><ul><li>Atomic nuclei – tiny magnets </li></ul></ul></ul><ul><ul><ul><li>In external field – align or oppose </li></ul></ul></ul><ul><li>Number of nucleons </li></ul><ul><ul><ul><li>Must be odd for residual magnetism </li></ul></ul></ul><ul><ul><ul><li>For e.g. Hydrogen exhibits, Deuterium does not </li></ul></ul></ul>Contd…
  7. 7. <ul><li>Resonance condition </li></ul><ul><ul><ul><li>Transition from low to high energy state occurs </li></ul></ul></ul><ul><ul><ul><li>h ﬠ = gßH </li></ul></ul></ul><ul><ul><ul><li>Either a constant magnetic field is applied and appropriate region of RF is swept </li></ul></ul></ul><ul><ul><ul><li>Or a constant frequency is employed & the external magnetic field is swept </li></ul></ul></ul>basic principle in NMR spectroscopy Contd…
  8. 8. <ul><li>Chemical shift </li></ul><ul><ul><ul><li>Arises from applied field inducing secondary fields at the proton by interacting with adjacent bonding electrons </li></ul></ul></ul><ul><ul><ul><li>Is measured relative to an internal standard – TMS </li></ul></ul></ul><ul><li>Spin – spin splitting </li></ul><ul><ul><ul><li>Seen in high resolution spectroscopy </li></ul></ul></ul>basic principle in NMR spectroscopy Contd…
  9. 9. <ul><li>E.g. of ethanol </li></ul><ul><li>a) Low resolution spectrum b) High resolution spectrum </li></ul>Contd…
  10. 10. Instrumentation
  11. 11. Diagram of NMR unit Contd…
  12. 12. instrumentation Contd…
  13. 13. Interpretation of NMR Spectrum <ul><li>Number of main signal = number of equivalent protons in unknown compound </li></ul><ul><li>Chemical shift indicates the type of H atoms. E.g. methylene, methyl groups, etc. </li></ul><ul><li>Spin spin splitting ---- arrangement of groups in the molecule. </li></ul><ul><li>Area of peaks ---- no. of H nuclei present in each group. For e.g. relative areas of methyl peaks in propane would be 6:2. In butane it would be 6:4 </li></ul>
  14. 14. E.g. of Ethanol
  15. 15. Application in the field of Biology
  16. 16. Application in the field of Biology <ul><li>Molecular structure determination </li></ul><ul><ul><ul><li>Most work done in solutions. </li></ul></ul></ul><ul><ul><ul><li>Drug metabolism studies & mechanism of drug action. </li></ul></ul></ul><ul><ul><ul><li>Low resolution NMR on lac repressor headpiece and BPTI. </li></ul></ul></ul><ul><ul><ul><li>High resolution NMR spectrum on structure of BDS-I, C3a & C5a inflammatory proteins, plastocyanin, thioredoxin, epidermal growth factor & interleukines </li></ul></ul></ul><ul><ul><ul><li>Solid state NMR --- Alzheimer’s B-Amyloid peptide. </li></ul></ul></ul>Contd…
  17. 17. E.g. of Ibuprofen
  18. 18. <ul><li>Molecular behavior in solutions </li></ul><ul><ul><ul><li>Results of studies of protein folding are exemplified by ribonuclease A, cytochrome c, barnase, lysozyme, ubiquitin and BPTI. </li></ul></ul></ul><ul><ul><ul><li>Enzyme kinetics, e.g. : chymotrypsin, trypsin, papain, pepsin, ATPase, ribonuclease, pyruvate kinase, etc. </li></ul></ul></ul>Contd…
  19. 19. <ul><li>Phosphate metabolism </li></ul><ul><ul><li>31 P isotope exhibits nuclear resonance </li></ul></ul><ul><ul><li>Changing concentration of AMP, ADP & ATP in living cells </li></ul></ul><ul><ul><li>Intracellular & extracellular Pi concentrations </li></ul></ul>Contd…
  20. 20. <ul><li>Nucleic acids </li></ul><ul><ul><li>Structural studies of DNA & RNA </li></ul></ul><ul><ul><li>Drug interactions with DNA </li></ul></ul><ul><ul><li>Interactions between binding proteins & DNA </li></ul></ul><ul><ul><li>Interaction between protein & lipid bilayers in membranes have been observed. </li></ul></ul>Contd…
  21. 21. <ul><ul><li>Magnetic Resonance Imaging (MRI) </li></ul></ul><ul><ul><ul><li>Resonance of 1 H is considered. </li></ul></ul></ul><ul><ul><ul><li>Proton is more sensitive. </li></ul></ul></ul><ul><ul><ul><li>Present in abundance . </li></ul></ul></ul>Contd…
  22. 22. <ul><li>Human brain </li></ul><ul><ul><li>Imaging of posterior fossae, brain stem, spinal cord </li></ul></ul><ul><ul><li>Detection of demyelinated lesions, hemorrhages. </li></ul></ul><ul><ul><li>Differences in water content in white and gray matter in brain & between normal tissues and tumors. </li></ul></ul>Contd…
  23. 23. <ul><li>Abdomen </li></ul><ul><li>Metabolic liver disease. </li></ul><ul><li>In adipose tissue --- proton signals from lipids is measurable & hence the fatty deposits can be viewed. </li></ul>Contd…
  24. 24. <ul><li>Advantages of MRI </li></ul><ul><ul><li>Eliminates risk of X-radiation. </li></ul></ul><ul><ul><li>Excellent and contrast resolution. </li></ul></ul><ul><ul><li>Detecting disease at earlier stages. </li></ul></ul>
  25. 25. Advantages of NMR spectroscopy <ul><ul><ul><li>A considerable amount of information can be obtained from a single analysis </li></ul></ul></ul><ul><ul><ul><li>The technique is non destructive </li></ul></ul></ul><ul><ul><ul><li>Replicate analysis may be performed </li></ul></ul></ul><ul><ul><ul><li>Precious samples may be recovered </li></ul></ul></ul>
  26. 26. References <ul><li>Principles and techniques in Biochemistry & Molecular biology, by Keith Wilson & Walker, 8 th edition. </li></ul><ul><li>Nuclear Chemistry by R. K. Dave </li></ul><ul><li>Biochemistry with clinical correlation by Thomas M. Delvin </li></ul><ul><li>Websites: </li></ul><ul><ul><li>http://biologyreference.com </li></ul></ul><ul><ul><li>http://chemnmr.colorado.edu/microinfo </li></ul></ul>