• 7.1 CALIBRATING A PULSE ON THE OBSERVE CHANNEL
• 7.1.1 Setting Up the Calibration Experiment
• 7.1.2 Setting up the Pulse Width Array
• 7.1.3 Calibrating Pulse Widths in H2O

• 7.2 CALIBRATING X NUCLEUS PULSES ON THE DECOUPLER CHANNEL
• 7.2.1 Calibrating the First Decoupler Channel
• 7.2.2 Calibrating C13 Pulses on the Second Decoupler Channel

• 7.3 CALIBRATING A SELECTIVE PULSE ON WATER
• 7.4 VALIDITY OF PULSE CALIBRATIONS

This section and those that follow deal with setting up NMR experiments. Wherever possible the guidelines tell you how to set up the experiment from scratch. As an alternative you can load one of the setup files from the uclib directory. In this case you will only need to adjust a limited subset of parameters. The name of the relevent setup file (if it exists) is given at the beginning of each section

Pulses are normally calibrated using the pulse sequence s2pul. For calibrating proton pulses in 90% H₂O the program presat should be used. The strategy is to acquire a 1D spectrum using a pulse length less than a 90[ring] pulse. The resulting spectrum is phased and acts as a reference for the calibration. Pulses than produce a net rotation less than 180[ring] will have positive intensities whilst those than produce rotations between 180[ring] and 360[ring] will have negative intensity. An exact 180[ring] or 360[ring] pulse produces no net signal intensity.

uclib: H1_calibration_D2O.fid
- Enter the command "s2pul", this sets up the parameters for the s2pul sequence,
- Set the transmitter offset (tof) to the appropriate value (Section 5.1)
- Set tpwr=60 (For P31 set tpwr =55) pw=4, d1=5, np=4096, fn=4096, nt=1, ss=4
- Enter the command "go"
- This acquires a single scan spectrum to use as a reference
- When the experiment has finished (20-30 secs) FT the FID and phase the spectrum

- Typical 90[ring] pulse widths are 5-10µs for 1H, 10-15µs for C13 and 15-20µs for P31.
- Set up an array to bracket the 360[ring] pulse width. e.g. for H1 type in the command "array" and answer the questions in the following fashion:
Parameter to be arrayed? pw
Number of steps 11
Starting Value 20
Enter array increment 2
- Then type "go"
- When the experiment has finished type " ft dssh"
- You should see a series of spectra display horizontally across the screen
- Adjust the parameter vs (vertical scale) and vp(vertical position) to get an optimal display of the spectra. If necessary adjust the display to show just a small region of the spectrum
- The display should look like Figure 7.1:

Figure 7.1 Typical output from a pulse array
- The 360[ring] pulse is the spectrum with the minimum intensity. In this example this is somewhere between points 5 and 6 (i.e. between 28 and 30 µs). The values used in the experiment can be displayed using the "da" (display array) command
- If necessary adjust the range of the array to obtain a final result similar to the one above. You can also cut down the range of the array to get a more accurate measurement of the 360[ring] pulse width
- When you have finished calibrating your pulse width, you can set pw to a 90[ring] pulse.

uclib: H1_calibration_H2O.fid
- The calibration is exactly the same except use the following commands:
- Enter "presat" to set up the parameters for solvent suppression.
- Set tof as above:
- Set satmode='y', satpwr=10, presat=1.5 and satfreq=tof
- Set composit and hs to 'n'
- otherwise proceed as above.

Many modern NMR experiments use samples that have been labelled with C13/N15. These experiments involve applying X pulses and X decoupling in addition to the pulses on 1H. In these cases you need to calibrate the X nucleus pulse using the same configuration that you will use for the experiment. The larger spectral widths of C13 experiments place a greater demand on the power requirements for applying RF pulses. The first decoupler channel is routed through the Magnet-Console interface. The presence of relays and filters within the interface leads to a loss in 2-3 dB of power compared to the second decoupler channel which is routed directly from the power amplifier into the probe. Consequently the spectrometers are normally configured to apply N15 pulses on the first decoupler channel and C13 pulses on the second decoupler channel. Although if you wish to change this you can do so. In any case the calibration is most readily acheived using the HMQC pulse sequence. The experiment is initially set up as a 2D experiment but a simple set of commands changes this into a 1D that we can use to calibrate the X nucleus pulses:

uclib: N15_calibration_HMQC.fid, C13_calibration_HMQC.fid
1. Calibrate the 1H pulse as described above.
2. Join a new experiment.
3. Enter the command "hmqc"
Respond with a <RETURN> to the question about the C13 experiment
4. Set up the 1H Parameters:
- Set tn='H1' and set pw, tof, sw, and np as outlined above.
- Set ss=4 and nt=16
5. Change unwanted parameters (Removes 2D setup):
- Set ni=1, phase=1, mbond='n', null=0, sspul='n'
6. Set up Presaturation Parameters for samples in H₂O:
- Set satmode='y', presat=1.5, satpwr=10, satfrq=tof
7. Set up N15 or C13 parameters :
- set dn and dof to relevant values
- set dm='n'
- set homo='n'
- set pwxlvl=60
- set j=90 for N15 and j=130 for C13
- set pwx=40 for N15 and pwx=10 for C13 (These are initial estimates of the 90[ring] Pulse)
8. Enter the go command
9. Transform and phase the spectrum
10. Now set up an array for pwx:
- For N15 use values between 30 and 60 µs (You may want to cut this down later)
- For C13 use values between 5 and 20 µs
- Run the experiment. It will take several minutes. If necessary increase the number of scans to obtain good S/N ratios
- Transform the spectra and display the results using the command "dssh"
- The spectrum with the maximum intensity corresponds to the 90[ring] pulse on the X nucleus.
NOTE For C13 calibration you must judge the maximum from the signals at the high field end of the spectrum. i.e. 0-3 ppm. Do not judge the intensity from the signals in the center of the spectrum

uclib:C13_calibration_3RF.fid
1. Load the Parameter File
- From the uclib directory load the file "C13_calibration_3RF.fid"
2. Set up the 1H Parameters:
- Set tn='H1' and set pw, tof, sw, and np as outlined above.
- Set ss=4 and nt=16
3. Change unwanted parameters (Removes 2D setup):
- Set ni=1, phase=1, mbond='n', null=0, sspul='n'
4. Set up Presaturation Parameters for samples in H₂O:
- Set satmode='y', presat=1.5, satpwr=10, satfrq=tof
5. Set up N15 parameters :
- set dn=`N15' and dof to relevant values
- set dm='n'
- set homo='n'
6. Set `C13' Parameters
- set dn2=`C13' and dof2 to relevent values
- set dm2='n'
- set pwxlvl=60
- set j=130 for C13
- set pwx=10 for C13 (These are initial estimates of the 90[ring] Pulse)
8. Enter the go command
9. Transform and phase the spectrum
10. Now set up an array for pwx:
- For C13 use values between 5 and 20 µs
- Run the experiment. It will take several minutes. If necessary increase the number of scans to obtain good S/N ratios
- Transform the spectra and display the results using the command "dssh"
- The spectrum with the maximum intensity corresponds to the 90[ring] pulse on the X nucleus.

uclib:sh2pul_calibration.fid
In H₂O samples, the high concentration of the solvent (100 M) leads to an effect known as "radiation damping". If the water magnetization is not aligned along the +Z axis it precesses and induces a voltage in the receiver coil. This voltage has an associated magnetic field which acts like a selective pulse on the water signal. This "pulse" acts to return the magnetization back to the +Z axis. This means it is difficult to manipulate the water signal in a controlled fashion. However the "radiation damping" effect can be exploited as part of a solvent suppression routine known as FLIP BACK. This sequence uses selective pulses on the water to maintain its position along the Z axis.
When calibrating a selective pulse on the water the radiation damping effects make it difficult to determine the length of the 360[ring] pulse from the appearance of the spectrum. It is much easier to determine the 360[ring] pulse from the appearance of the FID.
The length of a selective pulse is normally limited by other constraints within a more complex pulse sequence. Consequently we set the length of the selective pulse to a fixed value and adjust the power of the transmitter to obtain the 360[ring] pulse. Subsequently we adjust the POWER level to obtain the 90[ring] pulse. For Proton only spectra the selective pulse can be 3000-5000 µs duration. For 1H/15N and 1H/C13 spectra the pulse should be 2000-2500 µs :
1. Set up the Selective Pulse Experiment
- Enter the command "sh2pul"
- Set pw to the required value (see above)
- Set pwpat=`gauss'
- Set d1=1.5, ss=8, nt=1
- Set gain=2
2. Set up an array for tpwr from 10-35 dB
3. Display the FIDs with the command dfsh(display FID shifted horizontally)
- The resulting series should look something like this:

The second "null" is the 360[ring] pulse. The 90[ring] pulse has the same duration as the 360[ring] pulse but the power level is four times lower. i.e. from the relationships in section 5.6.3 this is the value of tpwr -12 dB. Typical power levels for a 360[ring] pulse are 26-30 dB and so typically the power for the 90[ring] pulse is 14-18 dB. If the array does not give a perfect 360[ring] pulse always choose the value that is slightly longer than the 360[ring] pulse. You will adjust it more accurately in the flip-back experiments.

The 90deg pulse width is a direct function of the strength of the RF. If you change the power of the RF by changing the value of tpwr, then the length of the 90[ring] pulse will also change. Therefore it is important that you always use the same transmitter power in your experiments that you used to calibrate your pulse lengths. The Proton pulse widths are the most sensitive on the probes that we have available and may change dramatically depending on the sample, salt concentration and temperature. Consequently the Proton pulse widths should be recalibrated for every new sample as a matter of routine. Generally we have found that the pulse widths for other nuclei are relatively insensitive to large differences in sample conditions. Therefore after calibrating these pulse widths for the first time you can use this value for most other samples.

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