Pharmacy 325

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Pharmacy 325
Nuclear Magnetic Resonance (NMR)
Spectroscopy Dr. David Wishart Rm. 2123 Ph.
492-0383 david.wishart_at_ualberta.ca Hours anytime
after 4 pm
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Lecture Notes Available At

• http//redpoll.pharmacy.ualberta.ca
• http//www.pharmacy.ualberta.ca/pharm325/

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Electromagnetic Spectrum
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Electromagnetic Spectrum
Hz
1021 1018 1015 1012
109 106
l (nm)
10-3 1 200 500 106 109
1012
Visible
Cosmic
X-ray
Ultraviolet
Radio
Microwave
Infrared
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Different Types of NMR

• Electron Spin Resonance (ESR)
• 1-10 GHz (frequency) used in analyzing free
  radicals (unpaired electrons)
• Magnetic Resonance Imaging (MRI)
• 50-300 MHz (frequency) for diagnostic imaging of
  soft tissues (water detection)
• NMR Spectroscopy (MRS)
• 300-900 MHz (frequency) primarily used for
  compound ID and characterization

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NMR in Everyday Life
Magnetic Resonance Imaging
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NMR Spectroscopy
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Explaining NMR
UV/Vis spectroscopy
Sample
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Explaining NMR
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Principles of NMR

• Measures nuclear magnetism or changes in nuclear
  magnetism in a molecule
• NMR spectroscopy measures the absorption of light
  (radio waves) due to changes in nuclear spin
  orientation
• NMR only occurs when a sample is in a strong
  magnetic field
• Different nuclei absorb at different energies
  (frequencies)

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Protons (and other nucleons) Have Spin
Spin up Spin down
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Each Spinning Proton is Like a Mini-Magnet
Spin up Spin down
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Principles of NMR
N
N
hn
S
S
Low Energy High Energy
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Which Elements or Molecules are NMR Active?

• Any atom or element with an odd number of
  neutrons and/or an odd number of protons
• Any molecule with NMR active atoms
• 1H - 1 proton, no neutrons, AW 1
• 13C - 6 protons, 7 neutrons, AW 13
• 15N - 7 protons, 8 neutrons, AW 15
• 19F 9 protons, 10 neutrons, AW 19

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The NMR Equation
n gB/2p

• B magnetic field strength in Tesla (1 Tesla
  10,000 Gauss 1000 kitchen magnets)
• g magnetic ratio (characteristic of each
  nucleus, each atom in a molecule

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Bigger Magnets are Better
Increasing magnetic field strength
low frequency high frequency
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Different Isotopes Absorb at Different Frequencies
2H
15N
13C
19F
1H
30 MHz 50 MHz 125 MHz 480 MHz
500 MHz
low frequency high frequency
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Typical 1H NMR Spectrum
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NMR Units of Measurement

• Energies 10-6 eV
• Wavelength 30 cm - 100 cm
• Frequency 108 - 109 Hz
• Parts per million (d) 0 - 12 ppm (for 1H)

nobs - nref
x 106
ppm
nref
ppm is proportional to frequency
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IR vs. NMR
Absorbance
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NMR vs. IR

• NMR has narrower peaks relative to IR
• NMR yields far more information than IR
• NMR allows you to collect data on solids
  liquids but NOT gases
• NMR is more quantitative than IR or UV
• NMR samples are easier to prepare
• NMR is much less sensitive than IR or UV
• NMR spectrometers are very expensive

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A Modern NMR Instrument
Radio Wave Transceiver
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NMR Magnet
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NMR Magnet Cross-Section
Sample Bore
Cryogens
Magnet Coil
Magnet Legs
Probe
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An NMR Probe
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NMR Sample Probe Coil
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A Modern NMR Instrument
Radio Wave Transceiver
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FT NMR
Free Induction Decay
FT
NMR spectrum
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Fourier Transformation
iwt
F(w) f(t)e dt
Converts from units of time to units of frequency
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1H NMR Spectra Exhibit...

• Chemical Shifts (peaks at different frequencies
  or ppm values)
• Splitting Patterns (from spin coupling)
• Different Peak Intensities ( 1H)

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Chemical Shifts

• Key to the utility of NMR in chemistry
• Different 1H in different molecules exhibit
  different absorption frequencies
• Arise from the electron cloud effects of nearby
  atoms or bonds, which act as little magnets to
  shift absorption n up or down
• Mostly affected by electronegativity of
  neighbouring atoms or groups

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Characteristic Chemical Shifts
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Spin-Spin Coupling

• Many 1H NMR spectra exhibit peak splitting
  (doublets, triplets, quartets)
• This splitting arises from adjacent hydrogens
  (protons) which cause the absorption frequencies
  of the observed 1H to jump to different levels
• These energy jumps are quantized and the number
  of levels or splittings n 1 where n is the
  number of nearby 1Hs

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Spin-Spin Coupling
H
H
H
H
C - Y
C - CH
C - CH2
C - CH3
J
singlet doublet triplet
quartet
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Spin Coupling Intensities
1 1 1 1 2 1 1 3 3 1 1 4 6 4 1 1 5 10 10 5 1
3 3
2
1 1
1 1
1 1
Pascals Triangle
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NMR Peak Intensities
Y
Y
Y

C - CH
C - CH2
C - CH3
AUC 1 AUC 2 AUC 3
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Applications

• Determination of exact structure of drugs and
  drug metabolites - MOST POWERFUL METHOD KNOWN
• Detection/quantitation of impurities
• Detection of enantiomers (shift reagents)
• High throughput drug screening
• Analysis/deconvolution of liquid mixtures
• Water content measurement

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Metabonomics

• Analysis of blood, urine and other biofluid
  mixtures to quantify and identify metabolite
  changes
• Allows one to detect drug toxicity and even
  localize toxicity (for preclinical trials) in a
  non-invasive way
• Detection, identification and quantitation of
  primary and secondary drug metabolites

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Metabonomics
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Other Applications

• Clinical testing (detection of inborn errors of
  metabolism, cancer, diabetes, organic solvent
  poisoning, drugs of abuse, etc. etc.)
• Cholesterol and lipoprotein testing
• Chemical Shift Imaging (MRI MRS)
• Pharmaceutical Biotechnology (proteins, protein
  drugs, SAR by NMR)