TOCSY On The AM-4/500 With Water Suppression

General Tips

As for all 2D experiments, don't spin the sample. Maintaining a stable temperature is very important. Turn the VT on to ~2-3 degrees above the current room temperature. After the temperature has equilibrated, shim and tune the probe.

Set up

First measure the water offset and set O1 and O2 equal to this value. Because we pulse through the decoupler channel for this experiment, it is necessary to measure the 90 degree proton pulse from the decoupler. To do this, first flip the switch on the console from "NORMAL" to "REVERSE" mode, and set PR = H 1.
Use the pulse program DECPCAL.AU =D1 to measure the 90 degree pulse.
First, use the AS command to set the parameters as follows:
	D5 = 10 ms (power switching delay)
	S3 = 3H (power at which the 90 degree pulse will be measured)
	D1 = 1.5s (relaxation delay)
	P1 = 2.8us (this value will be varied to find the 90)
	RD = 0
	PW = 0
	NS = 1
	DS = 0
Use the AU command to execute the pulse program (it will automatically perform the EFP command) and go into EP mode and the water peak with the A (Additive phase correction) command. Check to see that your phasing is okay by typing AU again. The spectrum should be properly phased, if not, repeat the above steps. Next vary P1 by small increments (5 us) to find the 180 or 360 pulse (spectrum will be null). Divide the value by 2 or 4 to get the 90 pulse. You can also adjust your SW at this point if desired.

Now you are ready to begin setting up the TOCSY experiment. If you have a small molecule with positive NOE's (MW<500-600) use the DANTOC.AU=D1 pulse program, or if you have a larger molecule with negative NOE's, use the DANCTOC.AU=D1 pulse program.
NOTE AS OF 1/2/96: DANTOC.AU WAS TESTED AND THERE APPEARS TO BE AN ERROR IN THE PHASE CYCLING!! USE THIS PULSE PROGRAM AT YOUR OWN RISK! PERSONALLY I RECOMMEND USING DANCTOC.AU FOR ALL MOLECULES AT THIS TIME!!!AS
Use your measured P1 value to calculate your loop counter L1 to get an appropriate mixing time for the TOCSY experiment. The mixing time should be 0.1/JHH, but for molecules with 3 to 4 component spin systems mixing times of 50-100 msec are usually appropriate. Lean towards 100 msec if your spin systems are long and/or your coupling constants are small. If you are using the DANCTOC.AU pulse program, your loop counter should be: 

 	L1 = (mixing time - 1msec)/147.2*p1
Don't forget to express p1 in milliseconds!
Also for this experiement you need to calculate the delay for the clean TOCSY sequence as follows: 
 	D20 = 2.6*p1
Or, if you are using the DANTOC.AU pulse program, calculate your loop counter as follows: 
 	L1 = (mixing time - 1msec)/64.67*p1
Again, p1 should be in milliseconds.

Now you are ready to begin setting up the TOCSY experiment. If you have a small molecule with positive NOE's (MW<500-600) use the DANTOC.AU=D1 pulse program, or if you have a larger molecule with negative NOE's, use the DANCTOC.AU=D1 pulse program.
Now AS the appropriate experiment and set the parameters as follows:

	D2 = 30 ms
	S1 = 15H
	P8 = 25 us
	D8 = 250 us
	L0 = 6000
The above parameters control the DANTE water suppression. If the suppression is too high or low, adjust L0 to lenghten or decrease the saturation time, or increase or decrease the power, S1. Be careful of increasing the power too much! The parameters for the rest of the TOCSY experiment are as follows:
	D7 = 30 ms
	S2 = 3H
	P4 = 180 degree pulse measured
	P1 = 90 degree pulse measured
	D0 = 3 us
	P0 = 1 ms
	P3 = 90 degree pulse measured
	D20 = set as calculated above (only for DANCTOC.AU experiment)
	P5 = 90 degree pulse measured
	P6 = 180 degree pulse measured
	L1 = set as calculated above
	D5 = 5 us
	D3 = 5 us
	D4 = will be set later
	D6 = will be set later
	NE = 512 W (typically for 12 h experiment)
	IN = will be set later
	S4 = 60H
Next, we need to set up the 2D parameters, which can be displayed on the screen using the ST2D command. Set TD, SI, TD2, and SI2 equal to 2K. Check to make sure TD1 is 512W if you want to take 512 experiments. Set ND0 = 2.

Now we can finish setting the parameters which were skipped over before. Type DW to see what value it is set to (this value should be something like 90 and it is expressed in microseconds). Set IN=DW, but be careful because DW is expressed in microseconds and IN is set in seconds, so if DW = 90, you need to type in IN = 90u (the letter "u" stands for microseconds). Always change IN, NOT DW. Next check the value of DE by typing DE and hitting return. DE is also expressed in usec, and set D4 = DE - 5usec.
Next check the value of AQ, which is expressed in seconds and set D6 = AQ + 2msec. Typical values for D4 and D6 are around 125 usec and 186 msec respectively. Finally, set DS = 2, DR = 16 initial and 12 final, and set NS to a multiple of 16 (NS = 32 will take approximately 11-12 h).

The last thing that needs to be done before running the experiment is to set the receiver phase (PH9). The pulse programs DANTOC.TST and DANCTOC.TST have been created to allow you to calculate the appropriate phase. Set NS=1 and AU the appropriate experiment. Change the receiver gain if necessary and retype AU until your receiver gain is correctly set (PLEASE NOTE: RGA will not work! The receiver gain must be set manually). Now restore NS to your desired value and collect one full experiment (it should stop after one experiment). Phase the spectrum using the "A" command (additive phase correction) in the EP mode and type "M" to memorize the phasing. Type the command "TY PHC0" and note the value of the zero order phase correction. Use the following formula to calculate PH9: PH9 = 360-(PHC0+90), but do not use negative values of PH9. If PH9 turns out negative from the above formula, add 360 to get a positive PH9. Now, enter this value into the pulse program DANTOC.AU or DANCTOC.AU (whichever you are using). Type "EDIT pulse program name" and move the cursor down to the line that starts out "PH9= (360)". After the (360), enter your calculated value of PH9 so the line now reads something like: PH9= (360) 261. Type escX to save your changes and exit the editor. AU either DANTOC.AU or DANCTOC.AU and enter your desired .SER=D1. DO NOT FORGET TO ADD THE .SER TO YOUR FILENAME. Watch the first scan of the first couple of experiments to make sure your receiver gain is okay. If everything looks fine, let 'er rip! Process on the X32 with MC2=TPPI, and zero order phase correction in F2 should be unecessary if you set the receiver phase properly.

Acknowledgment: This pulse program was written by Rachel Maplestone.
P.S. The AM500 has a tendency to crash if you leave the instrument in the job where the 2D experiment is running. As a precaution, move to a different experiment before leaving.
In addtion to these remarks, comments can be found at the bottom of the .AU programs.

Questions, comments to: Andrea Sefler andreafire.cchem.berkeley.edu

Last Update: 9/27/95.