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NMR Spectroscopy - PowerPoint


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									NMR Spectroscopy

     Part II. Signals of NMR
Free Induction Decay (FID)
       • FID represents the time-domain
         response of the spin system following
         application of an radio-frequency pulse.
       • With one magnetization at w0, receiver
         coil would see exponentially decaying
         signal. This decay is due to relaxation.
Fourier Transform
           The Fourier transform relates the
           time-domain f(t) data with the
           frequency-domain f(w) data.
Fourier Transform
Fourier Transform
NMR line shape

            Lorentzian line
                 W  4x0  x 
                    2                2

            A      amplitude
            W      half-line width

   Definition
    For signals in frequency domain it is the deviation of the
    peak line-shape from standard Lorentzian peak. For time
    domain signal, it is the deviation of FID from exponential
    decay. Resolution of NMR peaks is represented by the
    half-height width in Hz.
Resolution-digital resolution
   Measurement

half-height width:
            10~15% solution of 0-dichlorobenzene
            (ODCB) in acetone

              Chloroform in acetone
   Factors affect resolution

       Relaxation process of the observed nucleus
       Stability of B0 (shimming and deuterium locking)
       Probe (sample coil should be very close to the sample)
       Sample properties and its conditions
   Definition
    signal to noise-ratio

                 A     A:    height of the chosen peak
    s / n  2.5        Npp : peak to peak noise
                N pp
 Measurement
1H        0.1% ethyl benzene in deuterochloroform
13C       ASTM, mixture of 60% by volume deuterobenzene
            and dioxan or 10% ethyl benzene in chloroform
31P         1% trimehylphosphite in deuterobenzene
15N         90% dimethylformamide in deutero-dimethyl-
19F         0.1% trifluoroethanol in deuteroacetone
2H, 17O     tap water

   Factors affect sensitivity
       Probe: tuning, matching, size
       Dynamic range and ADC resolution
       Solubility of the sample in the chosen solvent
       Spectral Parameters
   Chemical Shift
    Caused by the magnetic shielding of the nuclei by their
    surroundings. d-values give the position of the signal relative to
    a reference compound signal.
   Spin-spin Coupling
    The interaction between neighboring nuclear dipoles leads to a
    fine structure. The strength of this interaction is defined as spin-
    spin coupling constant J.
   Intensity of the signal
Chemical Shift
   Origin of chemical shift

    Beff  B0  sB0  1  s B0
    s       shielding constant

                  
      '
           Beff     1  s B0
        2        2
    Chemically non-equivalent nuclei are shielded to different
    extents and give separate resonance signals in the spectrum
Chemical Shift
     Chemical Shift
   d – scale or abscissa scale
       1        1  s 1 
       2         1  s 2 
       2  1         s 2  s 1 
       2  1
                 s 2 s1
      Chemical shift parameterd  s 2  s 1  106
Chemical Shift
          d                     106
             observing frequency

                   Shielding s
                   CH3Br < CH2Br2 < CH3Br < TMS

                   d CHBr3  
                                            106  6.82 (ppm)
                                  90 106

90 MHz spectrum
Abscissa Scale
    Chemical Shift
   d is dimensionless expressed as the relative
    shift in parts per million ( ppm ).
   d is independent of the magnetic field
   d of proton           0 ~ 13 ppm
    d of carbon-13        0 ~ 220 ppm
    d of F-19             0 ~ 800 ppm
    d of P-31             0 ~ 300 ppm
        Chemical Shift
    s    s dia
                    s   local
                         para    s N s R s e si
   Charge density
   Neighboring group
        Ring current
        Electric field effect
        Intermolecular interaction (H-bonding & solvent)
       Chemical Shift –
       anisotropy of neighboring group

                               sN 
                                                     //    1  cos2 
                                          3r 3 4
                                      susceptibility
                                         r distance to the dipole’s center

Differential shielding of HA and HB in
the dipolar field of a magnetically
anisotropic neighboring group
  Chemical Shift –
  anisotropy of neighboring group

d~2.88               d~9-10
• Electronegative groups are "deshielding" and tend to move NMR signals from
  neighboring protons further "downfield" (to higher ppm values).
• Protons on oxygen or nitrogen have highly variable chemical shifts which are
  sensitive to concentration, solvent, temperature, etc.
• The -system of alkenes, aromatic compounds and carbonyls strongly deshield
  attached protons and move them "downfield" to higher ppm values.
•Electronegative groups are "deshielding" and tend to move NMR signals
from attached carbons further "downfield" (to higher ppm values).
•The -system of alkenes, aromatic compounds and carbonyls strongly
deshield C nuclei and move them "downfield" to higher ppm values.
•Carbonyl carbons are strongly deshielded and occur at very high ppm
values. Within this group, carboxylic acids and esters tend to have the
smaller values, while ketones and aldehydes have values 200.
    Ring Current
   The ring current is induced form the delocalized 
    electron in a magnetic field and generates an additional
    magnetic field. In the center of the arene ring this
    induced field in in the opposite direction t the external
    magnetic field.
Ring Current -- example
Spin-spin coupling
Spin-spin coupling
AX system
AX2 system
Spin-spin coupling
AX3 system
Multiplicity Rule
Multiplicity M (number of lines in a multiplet)
                  M = 2n I +1
n equivalent neighbor nuclei
I spin number
 For I= ½
Example   AX4 system
                I=1; n=3
         Order of Spectrum

Zero order spectrum
       only singlet
First order spectrum
        >> J
Higher order spectrum
        ~ J
AMX system
    Spin-spin coupling
   Hybridization of the atoms
   Bond angles and torsional angles
   Bond lengths
   Neighboring -bond
   Effects of neighboring electron lone-pairs
   Substituent effect
JH-H and Chemical Structure
   Geminal couplings 2J   (usually <0)

       H-C-H bond angle
       hybridization of the carbon atom
Geminal couplings J
                      bond angle
Geminal couplings J      2

 Substituent Effects   Effect of Neighboring
Vicinal couplings JH-H     3

     Torsional or dihedral angles
     Substituents
     HC-CH distance
     H-C-C bond angle
         Vicinal couplings JH-H
                                  dihedral angles

        Karplus curves

3      1 3
    J  2 J g 
                  
                 1 3
                     Jt 
         Shift of
         amino acid

   Chemical Shift Prediction

    Automated Protein Chemical Shift Prediction

 BMRB NMR-STAR Atom Table Generator for
    Amino Acid Chemical Shift Assignments
Example 1

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