Transcript Running NMR Experiment I

NMR Practical Aspects (I)
Processes involving in a NMR study:
1. Sample Preparation/Considerations
2. Setting up Spectrometer
3. Setting up acquisition parameters and carrying out experiment
4. Processing raw NMR fid data into NMR spectrum
5. Plotting of NMR Spectrum (1D with integrals of peak intensities)
6. Analysis of NMR data
References:
Basics of NMR: http://www.cis.rit.edu/htbooks/nmr
Practical: http://arrhenius.rider.edu/nmr/NMR_tutor/pages/nmr_tutor_home.html
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Safety Precautions: Very Strong Magnetic Field!
No Entry for Person with Heart Pace Maker
Keep and Secure
Ferromagnetic
Objects away from
the magnet
Aluminum or Stainless
Steel Ladder
10G
5G
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Sample Considerations:
•Molecular weight (MW):
Larger molecules => tumbling slower (c )
=> relax faster (i.e. shorter T2)
=> broader line-widths => lower sensitivities
MW
MW
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Sample Considerations:
•Concentration: relatively high conc. required because of low
sensitivity. At least ~0.1 mM for small organics; 15mM for protein or large macromolecules. For a given
S/N, half conc. takes four times longer expt. time.
•Solubility ( => S/N ), aggregation or polymerization (S/N ).
•Stability – tens of hours in the spectrometer may be required.
E.g. oxidation, microbial contamination, hydrolytic breakdown.
•Temperatures for data collection: T  => S/N 
need to consider the temperature dependence of stability
•Buffer/solvent selection: affect solubility, stability,
position of spectral absorption lines can be solvent
dependent, viscosity ( => S/N )
•Sample size: standard 5 mm tube need ~0.5ml sample volumn
other tubes to enhance sensitivity e.g. 10 mm tube, Shigemi tube
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Setting up Spectrometer procedures:
1. Insert Sample and set Temperature
2. Locking : Manual or Auto-locking
3. Tuning the probe: Manual or Auto-tuning
4. Shimming: Manual, Gradient shimming or Auto-shimming
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Setting up Spectrometer: Locking
Locking the deuterium signal from solvent to
maintain high magnetic field stability and high line
resolution
•Even in the best spectrometers the field strength
varies to some extent over time
•The position of the deuterium peak is monitored
• To counteract the field drift a lock field is
applied to maintain a constant deuterium
resonance position
Deuteriated solvent is usually used to provide
the Deuterium Lock signal e.g.
CDCl3, D2O, CD3OD
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Setting up Spectrometer: Tuning the Probe
• Variations in the polarity and dielectric constant of the
solvent will affect the probe tuning.
•Tune each coil to be resonant at the Larmor frequency for
the corresponding nucleus: e.g. 1H, 13C, 15N
•Two capacitors (tune and match) are adjusted to achieve
maximum power transfer into and out of the probe
Tuning with ‘wobb’ curve
Expected freq of the selected nucleus
Align the minimum
to expected freq
to get best tune
Minimize reflected power
to get best match
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Setting up Spectrometer: Shimming
•The process of making the magnetic field
surrounding the sample as homogenous as possible
•A series of shim coils correct minor inhomogeneities
in the static magnetic field
•Good Shims => Sharp lines!
All the nuclei in the sample “feel”
the same magnetic field
•Poor Shims =>
Broad lines and poor line-shapes!
Variable magnetic field across the
sample – nuclei in different regions
of the sample will resonate at
slightly different frequencies
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Setting up Spectrometer: Shimming
Shimming is judged at 50%, 0.55% and 0.11% of peak height in Hz
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Spinning Sidebands observed at multiples of spinning rate
What is the
spinning rate
here?
24 Hz
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No spinning for biomolecule samples because natural linewidth is large
Setting up acquisition parameters
For each dimension:
• Spectral Frequency (sf) : Center of all NMR peaks or H2O peak
position for aqueous sample
• Spectral Width (sw) in Hz : Covering all NMR peaks
• Number of data points (np) for direct detection dimension or
number of increments for indirect dimensions: Depends of
digital resolution required or experimental time available.
• Calibrate the 90 degree pulse width (pw90 in us) and set power
(in dB) for the pulses.
Global parameters:
• Recycling delay (d1) : >= 5T1
• Number of scans per fid (nt) : signal averaging to get better
signal to noise (S/N)
• Receiver Gain (rg) : maximize DAC usage
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Presaturation Experiment on protein Sample in H2O
tof is the transmitter offset frequency from
a base frequency e.g. 600.13 MHz
sf
sf+sw/2
[H2O]=55 M >> [protein]=1mM
> DAC resolution (e.g. 65535)
=> can’t see weak protein signals
sf-sw/2
sw
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Some useful terminologies and relationships:
Sampling rate: sr = 1/(dt)
in Hz; dt=dwell time
Optimal sampling rate is based on Nyquist criterion => sw = sr / 2
i.e. highest observable frequency = sample rate / 2
1
or dt = 1/(2*sw) in second
0.5
Acquisition time:
0.05
0.1
0.15
0.2
aq = dt * np
-0.5
How to detect aliasing peaks?
Alter sw
-1
These two frequencies are 10 and 110 Hz sampled at
Dt=0.01 seconds. The FT would have a SW of 100 Hz, and
the two peaks are indistinguishable. This is called aliasing
13 careful.
and can be a big experimental problem if you are not
Digital Resolution:
dr = sw / np
in Hz should be < natural line width
Improve dr either by  np or  sw
line width (lw)= 1/( T2)
Larger for bigger molecules
lw < 1 Hz for most organics
10-30 Hz for proteins
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Relationship between pulse width, power and Bandwidth
FT
Bandwidth (bw) = 1/(4 * pw) should be > spectral width
Power  6dB => pw90  by a factor of 2
Therefore can increase power until pw90 gives large enough 17bw
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3D data will requires increments on
ni and ni2 dimensions
i.e. ni * ni2 FID’s
Total time is in the order of 10 to 100 hours
Therefore the actual number of
increments for indirect dimensions is
usually a compromise between digital
resolution required and experimental
time available.
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Optimal Recycling Delay:
For full relaxation and restoration of equilibrium Magnetization
Recycling delay (d1) >= 5T1
But T1 can be very long for some nucleus e.g. 13C
=> very long acquisition time for full relaxation
t1
And signal averaging gives S/N 
Therefore one can get best use of experimental time (Texp) to
get best S/N by using the Ernst angle (e) for excitation in 1D
experiment:
e
cos e = exp(-Texp/ T1)
t1
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