(A PDF version of this handout
is available from the Handouts page or by clicking Here)
NMR experiments rely on 90 degree proton pulses for optimal performance
(e.g. HSQC, NOESY). The default values that are determined
by the facility may not be optimal for your sample because of differences
in solvent, salt concentration, etc. Therefore, you will need to
determine its value when you want to maximize the
of certain experiments. This page will guide you through the procedure
for calibrating a 90 degree pulse. Typically, you will look for the
360 pulse, which is a null spectrum, and then divide that value by
4 to get the 90.
Figure 1. Example of a pulse width calibration
experiment. The pulse width was arrayed from 2 microsecs to 40 microsecs
in steps of 2. Positive maxima occur at 90 and 450 degrees. The spectra with
almost no signal are the 180 and 360 degree pulse spectra. The 360-degree
spectrum would be used to calculate the 90-degree pulse width.
Note: Bold text represent
boxes you should click. italic text represent text you should type
and hit RETURN.
- Acquire a single scan proton spectrum (nt=1): transform
and phase. If S/N is poor, you can increase the number of scans (nt).
- Expand around any well resolved peak(s).
- Type d1=5. This will set the recycle delay
to 5 seconds. Complete relaxation between scans is necessary to get
- Arraying pw to find 360-degree pulse:
- Typically, you will look for the 360-degree pulse and
then divide the value by 4. The 360-degree pulse will give a null peak.
The total area (integral) should be zero. This is more accurate than
directly looking for the 90-degree pulse.
- Type pw90?. This will give the default 90 degree pulse
and is a good place to start.
- Type array. A set of questions will come up
and must be answered.
- Question#1: Parameter to be arrayed:
- Question#2: Number of increments:
- This is the
total number of spectra or data points you want. Typically, type
- Question#3: Enter Starting Value:
- Start with (4*pw90 - 1). For a pw90 of 10, I
would enter the value from (4*10-1) or 39.
- Quesiton#4: Enter Array increment:
- Type 0.5. This will increase the pw by
0.5 microseconds between each array element.
- Type pw=3. This will replace the first increment
with the value of 3, which will be positive.
- Type gain='y'
- Type go
- Type ai
- Type wft dssh. If the spectra are too small
to see, type vp=vp*2 dssh. Repeat command, if necessary. The
first spectrum (leftmost spectrum) should be positive. If it is not
- Type ds(1)
- Type f full. Phase the spectrum so that
the peaks are positive (aph usually works). Expand around
- Type dssh dssl. Look for the spectrum
with the minimum signal and note its number. If all the spectra
are positive, the starting value for pw was too large. Rerun the
array with a lower starting pw. Thus, in this case, I would start
with 37 instead of 39. If, on the other hand,
the second peak on are negative, the starting value of pw was too
small. Start with a larger value.
- Once you find the spectrum with the correct 360-pulse,
type da to list the array and find the pw that corresponds to the
spectrum (dssl lists the array number next to the spectrum).
- Type pw=your 360-degree pulse/4. For
example, I found the 360-degree pulse to be 38; therefore I typed pw=38/4.
- Type pw? and record the value. This is
the 90-degree pulse.
- Use this pulse width with your advanced experiment
(e.g. NOESY, HSQC, gHMQC)