Nmr Spectroscopy In B Iology
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Nmr Spectroscopy In B Iology






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Nmr Spectroscopy In B Iology Nmr Spectroscopy In B Iology Presentation Transcript

    • Introduction
    • Basic principles in NMR spectroscopy
    • Instrumentation
    • Interpretation of NMR spectra
    • Applications in the field of biology
  • Introduction
  • introduction
    • History
        • First described and measured by Isidor Rabi in 1938.
        • 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.
        • The observation of resonance frequencies of a molecule allows a user to discover structural information about a molecule.
  • Basic Principles in NMR Spectroscopy
  • basic principle in NMR spectroscopy
    • Magnetic phenomena
        • Atomic nuclei – tiny magnets
        • In external field – align or oppose
    • Number of nucleons
        • Must be odd for residual magnetism
        • For e.g. Hydrogen exhibits, Deuterium does not
    • Resonance condition
        • Transition from low to high energy state occurs
        • h ﬠ = gßH
        • Either a constant magnetic field is applied and appropriate region of RF is swept
        • Or a constant frequency is employed & the external magnetic field is swept
    basic principle in NMR spectroscopy Contd…
    • Chemical shift
        • Arises from applied field inducing secondary fields at the proton by interacting with adjacent bonding electrons
        • Is measured relative to an internal standard – TMS
    • Spin – spin splitting
        • Seen in high resolution spectroscopy
    basic principle in NMR spectroscopy Contd…
    • E.g. of ethanol
    • a) Low resolution spectrum b) High resolution spectrum
  • Instrumentation
  • Diagram of NMR unit Contd…
  • instrumentation Contd…
  • Interpretation of NMR Spectrum
    • Number of main signal = number of equivalent protons in unknown compound
    • Chemical shift indicates the type of H atoms. E.g. methylene, methyl groups, etc.
    • Spin spin splitting ---- arrangement of groups in the molecule.
    • 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
  • E.g. of Ethanol
  • Application in the field of Biology
  • Application in the field of Biology
    • Molecular structure determination
        • Most work done in solutions.
        • Drug metabolism studies & mechanism of drug action.
        • Low resolution NMR on lac repressor headpiece and BPTI.
        • High resolution NMR spectrum on structure of BDS-I, C3a & C5a inflammatory proteins, plastocyanin, thioredoxin, epidermal growth factor & interleukines
        • Solid state NMR --- Alzheimer’s B-Amyloid peptide.
  • E.g. of Ibuprofen
    • Molecular behavior in solutions
        • Results of studies of protein folding are exemplified by ribonuclease A, cytochrome c, barnase, lysozyme, ubiquitin and BPTI.
        • Enzyme kinetics, e.g. : chymotrypsin, trypsin, papain, pepsin, ATPase, ribonuclease, pyruvate kinase, etc.
    • Phosphate metabolism
      • 31 P isotope exhibits nuclear resonance
      • Changing concentration of AMP, ADP & ATP in living cells
      • Intracellular & extracellular Pi concentrations
    • Nucleic acids
      • Structural studies of DNA & RNA
      • Drug interactions with DNA
      • Interactions between binding proteins & DNA
      • Interaction between protein & lipid bilayers in membranes have been observed.
      • Magnetic Resonance Imaging (MRI)
        • Resonance of 1 H is considered.
        • Proton is more sensitive.
        • Present in abundance .
    • Human brain
      • Imaging of posterior fossae, brain stem, spinal cord
      • Detection of demyelinated lesions, hemorrhages.
      • Differences in water content in white and gray matter in brain & between normal tissues and tumors.
    • Abdomen
    • Metabolic liver disease.
    • In adipose tissue --- proton signals from lipids is measurable & hence the fatty deposits can be viewed.
    • Advantages of MRI
      • Eliminates risk of X-radiation.
      • Excellent and contrast resolution.
      • Detecting disease at earlier stages.
  • Advantages of NMR spectroscopy
        • A considerable amount of information can be obtained from a single analysis
        • The technique is non destructive
        • Replicate analysis may be performed
        • Precious samples may be recovered
  • References
    • Principles and techniques in Biochemistry & Molecular biology, by Keith Wilson & Walker, 8 th edition.
    • Nuclear Chemistry by R. K. Dave
    • Biochemistry with clinical correlation by Thomas M. Delvin
    • Websites:
      • http://biologyreference.com
      • http://chemnmr.colorado.edu/microinfo