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Validation of NMR Spectra

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Determine Quantity of a Substance. Relative and absolute amounts. Determine Purity of a Substance ... 1H NMR Spectrum of Ethyl Acetate. Relaxation Times: T1 and T2 ... – PowerPoint PPT presentation

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Title: Validation of NMR Spectra


1
Validation of NMR Spectra
  • William M. Egan, Ph.D.
  • Deputy Director
  • Office of Vaccines Research and Review
  • CBER/FDA

2
NMR Spectroscopy Primary Uses
  • Determine Structure
  • Primary structure
  • Higher order structure
  • Identity Test
  • Determine Quantity of a Substance
  • Relative and absolute amounts
  • Determine Purity of a Substance
  • Impurity profiles (limited)

3
The NMR Spectral Parameters
  • Chemical Shift
  • Spin-spin (scalar) Couplings
  • Integrated Absorption Intensity
  • Relaxation Times (T1 T2)

4
The NMR Parameters
  • Chemical Shift Resonance frequency is a
    function of the electronic environment
  • Integrated Absorption Intensity Is a function
    solely of the number of chemically equivalent
    nuclei giving rise to the signal (e 1 for all
    nuclei of a given isotope, e.g., all 1H or all
    13C)
  • Relaxation Times Are a function of relaxation
    mechanism and strength of the interaction (e.g.,
    dipolar coupling and internuclear distances) and
    motional behavior of the molecule (overall and
    internal motions correlation times)

5
1H NMR Spectrum of Ethyl Acetate
6
Relaxation Times T1 and T2
  • T1 Time constant for nuclei to come to their
    equilibrium distribution along the magnetic field
    axis, Bo. At equilibrium there is a net nuclear
    magnetism, Mo Mz.
  • T2 Time constant for nuclei to come to their
    equilibrium distribution perpendicular to the
    magnetic field. At equilibrium, there is no net
    nuclear magnetism in the x,y plane, Mx My 0.

7
Signal to Noise Ratio
  • Signal to noise ratio (S/N) is a function of the
    number of scans, n, that are collected
  • S/N increases as (n)½

8
Acquisition and Analysis of NMR Spectra
  • Spectral Analysis Carried out in the frequency
    domain, F(?).
  • Spectral Acquisition Carried out in the time
    domain, f(t), as the system response to a
    impulse.
  • Fourier Transformation Connects the time and
    frequency domains.

9
Fourier Transforms
  • F(?) I f(t) exp (-i?t) dt
  • f(t) (2p)-1 I F(?) exp (-i?t) d?
  • The integrals are evaluated from 4 to 4

10
Time and Frequency Domains
  • Frequency Domain
  • Chemical Shift
  • Integrated absorption intensity
  • Line width
  • Time Domain
  • Periodicity of signal
  • Signal intensity at time 0
  • Rate of signal decay

The Fourier transformation of an exponentially
decaying signal in the time domain is a
Lorentzian signal in the frequency domain
11
Relationship Between Time and Frequency Domains
Intensity of FID at t 0 proportional to
integrated absorption intensity in frequency
domain
Rate of decay of the FID defines the line width
in the frequency domain
Periodicity of FID defines the chemical shift in
the frequency domain
12
The NMR Spectrometer and the Assay
  • Is the NMR spectrometer functioning properly?
  • Number of tests to measure proper performance
    (S/N standards, etc.)
  • Is the assay measuring what is intended?
  • What can go wrong?

13
NMR and Magnetic Field Strength
  • Signal to noise ratio is affected by B0
  • The equilibrium magnetization, M0, is
    approximately linearly dependent on B0
  • The appearance of the spectrum is field dependent

14
Obtaining Quantitative Data
  • Assign particular resonances in the NMR spectrum
    to particular nuclei of the analyte, e.g., a
    particular methyl group
  • Obtain integral of resonance in the analyte
    relative to an assigned signal from a known
    amount of a standard added to the sample (e.g.,
    use D2O containing a known amount of DMSO).

15
Obtaining Quantitative Data
  • Magnetization must be at equilibrium in the
    magnetic field prior to each observing pulse
    f(T1)
  • No nuclear Overhauser effects (NOE) or,
    minimally, no differential NOE. The NOE is the
    change in z-magnetization at a nucleus, i, that
    results upon irradiation of nucleus, j.

16
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17
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18
Ruggedness/Robustness
  • Chemical shifts may be affected by a variety of
    factors, including
  • Concentration
  • Temperature
  • pH
  • Need to evaluate their influence
  • Shifting resonances could lead to unwanted signal
    overlaps, making the analysis difficult

19
W. Egan, Development Biologicals, 103, 3 9
(2000)
20
Lemercinier et al., Biologicals, 28, 17 24
(2000)
21
Archiving NMR Data
  • Need to archive the unprocessed Free Induction
    Decay (FID) signal.
  • Need to archive the FID meta-data (the parameters
    used to collect the FID).
  • Need to archive the processing parameters used to
    Fourier transform the FID to obtain the frequency
    domain spectrum from which information was
    extracted.
  • Not absolutely necessary to archive the frequency
    domain spectrum, since this can be recreated from
    the FID and the processing parameters (ZF, LB,
    etc.).
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