To begin, download the NMR pulse programs to the directory on the computer operating the NMR spectrometer and adjust the TopSpin version. Download the NMR shaped pulses to the directory and verify that the TopSpin version is updated accordingly. After determining the spectral widths and appropriate acquisition times, type edc in the TopSpin software to a new directory to copy the HSQC experiment.
To open the pulse sequence parameters type ased, click on the three dots next to the PULPROG parameter to change the pulse program to nitrogen-15 R1 rho experiment. Double-click on the pulse program, click on Set PULPROG to the dataset and confirm with OK.Type ased to open the pulse sequence parameters. Enter all missing gradient strengths and gradient lengths as given in the pulse sequence.
Next, enter for the loop counter L3 half of the nitrogen-15 TD entry in the acquisition parameter window. Enter for the loop counter L6 the number of recorded relaxation data points for fitting the relaxation curve. Set the adiabatic TanhTan_half.
nl pulse as the shape of the nitrogen-15 SP8 pulse. Then set the adiabatic TanhTan_half2nd. nl pulse as the shape of the nitrogen-15 SP9 pulse.
Ensure the pulse lengths are sufficiently long for adiabaticity with P8 set to 3, 000 microseconds. Then set the interscan recovery delay, D1, to at least two seconds or longer. Set the dummy scans to at least 64.
As a starting point set the number of scans to four and use multiples of four if the signal-to-noise ratio is too low. Set the O1 to the calibrated carrier frequency, O2P to 176 parts per million, and copy the O3P from the proton nitrogen-15 HSQC experiment. Now, set the pulse length P7 to the 90-degree pulse length calibrated earlier.
Then copy the pulse power level of the 90-degree pulse to PLW3 and PLW7. Afterward, set the pulse lengths P1 and P19 to the 90-degree proton pulse length. Set the number of increments in the indirect dimension, TD equals L3 by two by L6.Next, set the shaped pulse SP5 to an I-BURP2 shape and the pulse length P15 to 2, 000 microseconds.
Then open the Shape Tool display by clicking the E next to the I-BURP2 shaped pulse in the TopSpin pulse sequence parameter window. To simulate the shaped pulse click the Start NMR Simulation button. Check the Shaped pulse length and the rotation angle in the simulation window and click on Start NMR SIM to proceed.
Check the excitation range in the simulation and select the appropriate I-BURP2 pulse length to cover the proton spectral dispersion while avoiding excitation of the water. Set P15 to the shaped pulse length from the simulation window with the best I-BURP2 pulse. Now, set SPOFFS5 to adjust the carrier frequency of the I-BURP2 pulse, shifting the excitation range left or right to avoid water magnetization disturbance.
Then open the Bruker Shape Tool and click Start NMR Simulation to determine the appropriate power level of the shaped pulse. Set the I-BURP2 pulse length to the shaped pulse length, and note the soft rectangular 90-degree proton pulse length shown in the simulation window. Type calcpowlev to calculate the power level difference in decibels between the hard 90-degree proton pulse and the soft rectangular 90-degree proton pulse.
Copy the power level of the hard 90-degree proton pulse to SPW5 and adjust by adding the memorized difference in decibels. To determine the power level of the spin-lock calculate the corresponding 90-degree nitrogen-15 pulse length. Use calcpowlev to calculate the power difference in decibels between the spin-lock power and the hard 90-degree nitrogen-15 pulse.
Copy the power level of the hard 90-degree nitrogen-15 pulse to PLW7 and adjust the spin-lock power level PLW8 by adding the calculated power difference. Copy the power level of the spin-lock PLW8 to the power levels SPW8 and SPW9. To determine the power level of the nitrogen-15 decoupling use calcpowlev to calculate the power difference between the power of the 90-degree nitrogen-15 decoupling pulse and the 90-degree nitrogen-15 hard pulse.
Copy the power level of the 90-degree nitrogen-15 hard pulse PLW7 to the decoupling power level PLW31 and adjust by adding the calculated power difference in decibels. For temperature compensation include line define TEMP_ in the pulse program. Set P18 to the maximum duration of the spin-lock used in the nitrogen-15 R1 rho experiment.
In the case of nitrogen-15, carbon-13-labeled samples include the line define LABEL_CN in the pulse program. Set P4 to the calculated pulse length. Use calcpowlev to calculate the power difference in decibels between P4 and the carbon-13 hard pulse.
Copy the power level of the carbon-13 hard pulse to PLW4 and add the calculated power difference in decibels. Copy PLW4 to PLW2. To determine the appropriate relaxation delays for sampling run the first eight free induction decays, or FIDs, and process them.
Choose vplist entries where the peak intensity of the longest delay experiment decreases to at least one over e, but not lower than 25%compared to the shortest delay experiment. Type rga in the command line to determine the receiver gain. Start a test run of the nitrogen-15 R1 rho experiment by typing zg in the command line.
Check that the water signal is suppressed for all delays. Also, check increment nine, the second increment of the quadrature detection scheme.
