¹H hard pulse
¹H selective pulse (water Flip Back, FB, pulse)
¹⁵N hard pulse (inverse mode)
¹³C hard pulse (inverse mode)
¹³C selective 90 pulse (shaped pulse)
¹³C selective inversion (180) pulse (shaped pulse)
²H decoupling pulse
Calibrated Pulse-Length Values

¹H hard pulse

Set the power level (pl1) to 0-3 dB (depending on the probe you are using). Type p1 and change the value for small pulse angles to give a small but positive signal (typically start with p1~3-4 µsec). Acquire and process the spectrum (zg, ft, pk) to give a positive signal. [Notice that when the phase correction is defined so that the signal is positive for small pulse angles, then the signal will be positive when the pulse angle is slightly less than 180° and will be negative when the pulse angle is slightly more than 180°]. Change p1, acquire and process the new spectrum (zg, fp), until the signal goes through a null, indicating a 180° pulse.

90deg    180deg

A convenient way to calibrate a 90° pulse is with the automation program paropt. To start the automation program, simply type paropt and answer the following:

• Enter parameter to modify: p1
• Enter initial parameter value: 16
• Enter parameter increment: 0.3
• Enter # of experiments: 10

In this case, paropt acquires and processes 10 spectra while incrementing the parameter p1 from 16 µsec to 19 µsec. For each value of p1, only the spectral region defined above (dp1 icon) is plotted. All 10 spectra appear side by side in FILENAME/1/999.
The 1H 90° pulses of the MLEV sequence used during the spinlock period of a TOCSY sequence should be 30 to 40 µsec, while the one required for the cw spinlock used during ROESY corresponds to a 90° pulse length of 100 to 120 µsec. In order to find the corresponding power levels, one can use paropt and modify the parameter pl1 (keeping p1 constant). Alternatively, the user may make use of the rule of thumb that the pulse length should double, approximately, for every 6 dB decrease in power level. 

¹H shaped pulse (water flip-back, FB, pulse)

The "water flip-back" is a technique that utilizes field gradients for water suppression by returning the water magnetization to the z-axis prior to acquisition. It uses a selective 90 degree pulse on the water, a shaped flip-back pulse.
 
On the AVIII800:

• Use the 1D pulse program "calib_fb_H.ts", which contains both a 1H 90 degree pulse
  { (p1 ph0): f1 } and the FB pulse { (p2:sp2 ph25:r): f1 }.
• First, comment out the FB, run 1D and phase the spectrum.
• Comment out the 1H 90 degree pulse and apply the FB. Calibrate a long (p2~ 1-2 msec) 1H shaped pulse (spname2 = sinc1.0) and determine its phase (PHCOR25).
• Apply both the 1H 90 hard pulse as well as the selective FB pulse. Optimize the power level of the FB pulse (sp2) so that zero signal will appear. Correct the phase (PHCOR25) of the FB to get the minimal signal.

 ¹⁵N hard pulse (inverse mode)

The 15N pulse calibration is done in the inverse mode (f1= 1H, f3= 15N), using the pulse sequence DECP90. During calibration, the length and/or strength of the 15N pulse is adjusted. When the 15N pulse is exactly 90°, the anti-phase signal (~5.4 ppm) is minimal. When using the calibration sample 15N-Urea & 13C-Methanol in DMSO, o1p= 5.4 ppm, o2p=76 ppm, p1<90 (~3µsec) and the delay 1/(^2J_XH)= 5.94 msec should be used. The data set UreaMeth 2/1 [in /opt/topspin/data/tali/nmr) is already set to calibrate the 90 hard pulse of 15N. Start with setting the correct phase (to get the anti-phase signal) using a low power 15N pulse, pl3=20 dB (or use a much shorter than desired p21value). Increase the powe level (decrease pl3 values; or, alternatively increase p21) till the anti-phase signal (~5.4 ppm) is minimal.

                    <90 deg                                   90 deg             >90 deg

¹³C hard pulse (inverse mode)

The 13C pulse calibration is done in the inverse mode (f1= 1H, f2= 13C), using the pulse sequence DECP90. During calibration, the length and/or strength of the 13C pulse is adjusted. When the 13C pulse is exactly 90°, the anti-phase signal (~3.2 ppm) is minimal. When using the calibration sample 15N-Urea & 13C-Methanol in DMSO, o1p= 3.12 ppm, o2p=49 ppm, p1<90 (~3µsec) and the delay 1/(2JXH)= 3.6 msec should be used. The data set UreaMeth 3/1 [in /opt/topspin/data/tali/nmr) is already set to calibrate the 90 hard pulse of 13C. Start with setting the correct phase (to get the anti-phase signal) using a low power 13C pulse, pl2~20 dB (or use a much shorter than desired p3 value). Increase the powe level (decrease pl2 values; or, alternatively increase p3) till the anti-phase signal (~3.12 ppm) is minimal.

Selective Hards Pulses: Off-Resonance Effects

Selective Excitation

In order to avoid excitation of frequencies with an offset dW (Hz), the on-resonance pulse duration for the 90 degree pulse will be
t₉₀ = √15 / 4 (ΔΩ )
where  ΔΩ = [basic frequency] * [chem. shift defference in ppm].

Selective Inversion

In order to avoid excitation of frequencies with an offset ΔΩ (Hz), the on-resonance pulse duration for the 180 degree pulse will be

t180 = √3 / 2 (ΔΩ)
and ΔΩ = [basic frequency] * [chem. shift defference in ppm]

¹³C selective 90 pulse (shaped pulse)

The selective 13C pulse calibration is done in the inverse mode (f1= 1H, f2= 13C), using the pulse sequence DECP90SP. During calibration, the length (p13 here) and/or strength (sp2 here) of the 13C pulse is adjusted. When the 13C pulse is exactly 90°, the anti-phase signal (~3.2 ppm) is minimal. When using the calibration sample 15N-Urea & 13C-Methanol in DMSO, o1p= 3.12 ppm, o2p=49 ppm, p1<90 (~3µsec) and the delay 1/(^2J _XH)= 3.6 msec should be used. Set the name of the selective pulse (SPNAM2 here) to g4.256. The data set
UreaMeth 4/1 [in /opt/topspin/data/tali/nmr]
is already set to calibrate the 90 selective pulse of 13C. Start with setting the correct phase (to get the anti-phase signal) using a low power 13C pulse, sp2~20 dB (or use a much shorter than desired p13 value). Increase the powe level (decrease sp2 values; or, alternatively increase p13) till the anti-phase signal (~3.18 ppm) is minimal

¹³C selective inversion (180) pulse (shaped pulse)

The selective 13C pulse calibration is done in the inverse mode (f1= 1H, f2= 13C), using the pulse sequence DEC180SP. During calibration, the length (p14 here) and/or strength (sp3 here) of the 13C pulse is adjusted. When the 13C pulse is exactly 180°, the in-phase signal (~3.2 ppm) is minimal. When using the calibration sample 15N-Urea & 13C-Methanol in DMSO, o1p= 3.12 ppm, o2p=49 ppm and the delay 1/(^2J_XH )= 3.6 msec should be used. Set the name of the selective pulse ( SPNAM2 here) to g3.256. Use the calibrated 90 hard pulse values for both 1H (p1) and 13C (p3), long d1 values (4-5 sec), and ns= ds =2. The data set UreaMeth 5/1 [in /opt/topspin/data/tali/nmr) is already set to calibrate the 180 selective pulse of 13C. Start with setting the correct phase (to get the doublet) using a low power 13C pulse, sp3~20 dB (or use a much shorter than desired p14 value). Increase the powe level (decrease sp3 values; or, alternatively increase p14) till the signal (~3.18 ppm) is minimal

²H decoupling pulse (AVIII800)

The use of partially and/or fully 2H labeled samples enables NMR studies of larger proteins in solution. In order to enable alternation of the lock channel between locking and decoupling during the experiment, a free rf-channel and a lock-switch are required. This hardware is available on the AVIII800 spectrometer. The sample used for calibrating the 2H decoupling pulse can be the standard ASTM sample (C₆D₆ in dioxane). The rf routing should enable 13C observation (F1), and 2H should appear on F4. The data set UreaMeth 6/1 [in /opt/topspin/data/tali/nmr) is already set to calibrate the 180 selective pulse of 13C.Use the pulse program decp902hf4, and set o1p=128.3 ppm and o4p=7.28 ppm. The experiment is the same as that used for 15N and 13C inverse 90 calibration (decp90), but is modified to include the 2H switch. At 90 degree, the signal turns into an antiphase that turns its sign when passing the 90.
 

Calibrated Pulse-Length Values

Calibrated Pulse-Length Values for various nuclei on AVIII800, AV-500, AVIII400 and AVIII300 probes.