Choosing an experiment

1
The basics of high resolution NMR

• Choosing an experiment
• Sample preparation
• Choosing a probe
• Choosing a pulse program
• Inserting the sample
• Tuning
• Shimming
• Calibrating pulses and entering starting
  parameters
• Running the experiment or groups of experiments
  

2
The basics of high resolution NMR

• Choosing an experiment (simpler is better)
• What is the question?
• Is your sample soluble?
• What solvent?
• In D2O or H2O?
• Observe 1H or other nucleus?
• 1D, 2D, or 3D?
• Sample preparation
• Choosing a probe
• Choosing a pulse program

3
The basics of high resolution NMR

• Choosing an experiment
• Sample preparation (is critical)
• In D2O or H2O (deuterium is needed to lock)
• 1 mM protein in 600 microliters (for 5 mm tube)
• Filter if necessary, no particulate material
• Higher concentrations are better for sensitivity
• Low salt is better for tuning
• Avoid buffers with protons (phosphate works
  fine)
• Avoid solvents with more than one resonance.
• Choosing a probe
• Choosing a pulse program

4
The basics of high resolution NMR

• Choosing an experiment
• Sample preparation
• Choosing a probe
• Direct observe 1H (13C, 15N indirect dimensions)
  TXI
• Direct observe 1H (plus one X dimension) BBI
• Direct observe other nucleus (e.g. 31P) BBO
• MAS probe
• Temperature range?
• Choosing a pulse program

5
The basics of high resolution NMR

• Choosing an experiment
• Sample preparation
• Choosing a probe
• Choosing a pulse program
• Basic pulse program - e.g. NOESY
• Presaturation with water?
• Other type of water suppression (e.g.
  watergate?)
• With gradients?
• Phase sensitive?
• With or without random variation of mixing time?

6
The basics of high resolution NMR

• Inserting the sample
• Wipe outside of tube.
• Set height in tube holder.
• Turn on air eject.
• Do not approach magnet with anything made of
  metal.
• Insert sample into bore of magnet.
• Turn off air eject.
• Tuning
• Shimming
• Calibrating pulses and entering starting
  parameters
• Running the experiment or groups of experiments
  

7
The basics of high resolution NMR

• Inserting the sample
• Tuning (and matching)
• Determine NMR frequencies needed
• Shimming
• Calibrating pulses and entering starting
  parameters
• Running the experiment or groups of experiments
  

8
The basics of high resolution NMR

• Inserting the sample
• Tuning
• Shimming (39 gradient coils)
• Autoshimming
• Gradient shimming
• Only on TXI probe
• Only samples in water
• Manual shimming
• Z, Z2, Z3, Y (major shims on 700)
• Calibrating pulses and entering starting
  parameters
• Running the experiment or groups of experiments
  

9
The basics of high resolution NMR

• Inserting the sample
• Tuning
• Shimming
• Calibrating pulses and entering starting
  parameters
• Determine which pulses need to be calibrated from
  pulse sequence.
• Usually just 90 pulse.
• Determine procedure for calibration.
• Paropt parameter.
• Linear amplifiers and digital electronics make
  life easy.
• Running the experiment or groups of experiments
  

10
The basics of high resolution NMR

• Inserting the sample
• Tuning
• Shimming
• Calibrating pulses and entering starting
  parameters
• Running the experiment or groups of experiments
  
• ZG zero go
• Multizg run multiple experiments back-to-back

11
Practical Considerations

• Sample preparation
• Molecular weight
• TROSY - large molecules
• ROESY - small molecules
• Partial deuteration
• b-sheet vs a-helix
• Aggregation and solubility
• Dilution
• Mutagenesis

12
Practical Considerations

• Calibrating pulses and entering starting
  parameters
• Number of t1 increments
• Dwell and spectral width
• Resolution
• Number of points
• Magnetic field
• Line broadening and zero filling
• Linear prediction
• Phase cycling
• Suppression of artifacts
• Selection of coherence transfer pathway

13
Dwell and spectral width
The Nyquist Theorem says that we have to sample
at least twice as fast as the highest frequency
signal.
If we sample at half the frequency of the
highest frequency signal, the signal will be
digitized and correspond to a frequency that is
half of the real frequency.
14
Dwell and spectral width
dw
sw
frequency
time
dw 1/sw
15
Dwell and spectral width
real peak
frequency
time
aliased peak
16
Line broadening
No smoothing of FID
Exponential line broadening function
Maximum smoothing of FID
17
Line broadening
LB 5.0 Hz
LB -1.0 Hz
FT
FT
Best signalnoise when LB equals the line width
w/o line broadening
18
Zero filling
8K data
8K zero-fill
8K data points
4K data points
Collect data until FID goes to zero. However,
you still may not be able to define the top of
the peak. Then zero fill.
19
Zero filling
frequency
Points from zero filling fall between the real
points and improve digital resolution.
frequency
20
Phase cycling
FT
w
FT
w
wo
DC offset
Often times the baseline is not zero, but there
is some current in the coil that leads to an
offset from zero. The FT of a step function is a
delta function at zero frequency.
21
Phase cycling
90x
FT
w
wo
DC offset
90-x
FT
w
wo
Change phase of pulse from x to -x, and the phase
of real peak inverts IF the receiver does not
change phase.
22
Phase cycling
90x
FT
w
wo
DC offset
90-x
FT
wo
w
Change phase of pulse from x to -x, and the phase
of the receiver from x to -x.
23
Phase cycling
wo

w
w
wo
wo
w
These sorts of artifacts can be eliminated by
phase cycling.
24
Phase Cycling
90 Pulse
Rcvr
Cycle
1
0 (x)
0 (x)
2
90 (y)
90 (y)
3
180 (-x)
180 (-x)
4
270 (-y)
270 (-y)
Cyclops phase cycling
25
Phase Cycling
90
90
90
p 2 p 1 p 0 p - 1 p - 2
Double quantum filtered COSY experiment. Only
double quantum coherences are observed. This
greatly reduces the intensity of the diagonal in
the 2D COSY spectrum. The double quantum
coherences are selected by the phase cycle.