February 15, 1992 R. Nunlist
The TOCSY (TOtal Correlation SpectroscopY) experiment is ordinarily used to map
spins belonging to a common system. This is accomplished by the application of
a spin lock pulse with a suitable mixing time to let magnetization propagate
through the mutual spin-spin couplings. If the mixing time is short, the total
correlation fails: All we get is a regular COSY - like spectrum. By itself,
this is nothing extraordinary; why not use the regular COSY experiment? The
answer is found in a paper by Marion (J. Magn Reson 85, 393, 1989). The TOCSY
experiment uses a 'trim' pulse which leads to rapid de-phasing of the
magnetization. This eliminates the need for phase cycling. By using the trim
pulse, we can therefore work with one scan per increment and reduce the recycle
delay to about T1!
By comparison, the COSY type experiments require a minimum phase cycle of four
steps, often more steps are used to further reduce artifacts. In addition, if
the recycle delay is shorter than 3 x T1, further problems can arise.
Using the TOCSY experiment with a short mixing time (10 to 12 ms), it is
possible to acquire a COSY equivalent at least four times faster. Of course,
it is assumed that the S/N is not the limiting factor. With most samples, this
is not a problem.
Below is an example of our old favorite, rotenone. About 5 mg of sample, using
the QNP probe, yielded this in about
6 minutes!
A mixing time of about 12 ms, L6 = 6 was used. With no dummy scans, and NS = 1,
77 increments were acquired before I ran out of time. Most time was spent
writing to disk - acquisition plus disk write averaged about 6 seconds on the AM-400.
The VBAMX should produce the same result in less than 2 minutes - the disk write
takes only 30 ms!
Assuming the system is shimmed and locked, determine O1 for regular proton nmr
(default or use SO). Then, set O2 to that value.
Set PR = H 1 (this MUST be done).
Use AS INVH1.AU to set up the proton parameters.
Set NS = 1, P1 = 37, S1 = 6. AU to set the receiver gain.
Set P1 around 75, determine the 180 deg pulse (not very critical).
Set P1 to 1/2 of what you found the 180 deg pulse to be. S1 should already be set. Set P2 to 180 deg. Set P4 to 2000 (the 2 ms trim pulse). Set D1 to about T1. L6 should be set such that (L6 x 66 x P1) is about 10 to 12 ms. Set NE to 256. After the acquisition has finished, use NET (BRUKNET) from the second terminal to send the .SER file to the X32 workstation.
Size: 1k x 256w, zero filled to 512w x 512w final matrix Acquisition: 5 to 30 minutes, depending on NS, D1 and NE Processing: About 10 minutes on the X32 Plotting: 5 minutes
1) Use: RJ COSYDQB.500( or COSYDQQ.400)=D1 followed by PJ <cr><cr>, RJ2D COSYDQB.500. This sets default values for size, window functions etc. The width is set for 8 ppm to 0.25 ppm. (TMS will fold at 0.5 ppm!). For solvents other then CDCl3, use 'SO' to determine new O1, then set O2 to that value. If the window needs changing, use 'OW' to select new window. Note that after SO, SR1 and SR2 need to be changed to the same value as SR. If OW was used, 'ST2D' is needed again; now set I2D back to 1. Be sure that O2 and DW have been typed!
Set NS =1, DS=0.
If the 90 deg decoupler pulse is not known, determine it as follows:
- Set PR = H 1 (this MUST be done).
- Use AS INVH1.AU to set up the proton parameters.
- Set NS = 1, P1 = 37, S1 = 6. AU to set the receiver gain.
- Set P1 around 75, determine the 180 pulse by varying P1 (not very
critical).
- Set P1 to half the value of the 180 pulse.
Now proceed:
Set NE.
'WJ2D MYTOCSY.001' For later parameter print-out.
Set MAXY=CY=5. 'TI' if title is desired. Store spectrum on disk.
2)Be sure all parameters are set as follows:
F2 F1
TD= 1k 256w SI= 1k 1k SF= (SF of 1D) (SF of 1D) SW= (SW of 1D) (will be SW/2) HZ/PT= » 2 to 10 WDW= S S LB= i. i. GB= i. i. SSB= 2 2 HZ/PT2/HZ/PT1= 1 SIZE.SER IN K=256k REDF= N SIZE.SMX IN K REV= Y MC2= W MAXM= >500 NE= 256 ND0= 2set I2D = 1. (this resets SI1, Set SI1=1k again)
The SI, TD and NE values may need to be adjusted to obtain digital resolution of 2 to 6 Hz, depending on sweep width SW! This is best done by setting:
New SI, TD. Enter new NE (= SI/4 for square matrix). Type ST2D to update the relevant 2D parameters. Store the new parameters with 'WJ2D MYTOCSY.001'.
F2 F1
TD 1k TD1 256w Number of points to acquire SI 1k SI1 1k Number of points to transform SSB1= SSB2=2 90 ([[pi]] /2) shifted sine-bell WDW1= WDW2=S window for both dimensions MC2 = W for phase sensitive calculation REV = Y to reverse diagonal REDF = N ND0 = 2 Two D0's in pulse sequence. Set SI1 again to 1k I2 = 1 sets SW1 = SW/2 for square matrix NE = 256 number of increments (serial files)
3) Without exiting the parameter display, type 'AS MLEV17PH.AU' to set up the pulse program parameters.
PW = RD =0
P1 = 90 deg 37.5 usec @S1 = 6 (on AM-400)
P2 = 180 deg
P4 = 2000 (2 ms trim pulse)
P6 = 180 deg 75 usec @S1 = 6 (on AM-400)
D0 = 3E-6 (3 usec)
L6 = 5 (12 ms mix)
D1 = about T1. This depends on your sample!
NS = 1..
DS =0
IN =set by SW1
4) EXPT estimate time required. (Note: EXPT sums all delays
whether they are used or not. It may give an unreasonable
estimate if unused delays are not set to zero.)
5) AU File name will be asked for. MUST HAVE .SER extension, e.g.
MYTOCSY.SER.
While the 2D spectrum is acquired, the 1D FID can be sent to the X32 and
processed for use as projections. use NET (BRUKNET) from the second terminal
to send the FID, e.g. MYTOCSY.001=D3.6) After the end of acquisition:
Send the MYTOCSY.SER to the X32 for processing. If you insist to process on the instrument:
RJ COSYDQQ.400=D1, PJ <cr><cr>. ST2D MYTOCSY.SER=D2 (or D4). RJ2D MYTOCSY.001 (or COSYDQQ.400) to get parameters back. XFB [MYTOCSY.SER] to transform in both dimensions and generate MYTOCSY.SMXBefore processing, the phase parameters need to be set. The 1D phases should be used. If not noted above, switch jobs, PJ MYTOCSY1D.001. Type TY to read phase values. In 2D job, set PC0 = PHZ0-90deg. , PC1 = PHZ1. Execute phase correction with 'PZ'. These values will be used for phase correction after first FT.
Type ST2D [MYTOCSY.SER]. SET SI1 =SI2. Set, if needed: SR1 = SR2(1D), ND0=2, WDW1 = WDW2 = S, SSB1 = SSB2, = 2 ([[paragraph]] /2).
XFB [MYTOCSY.SER] to transform in both dimensions and generate MYTOCSY.SMX
View positive ('+') and negative levels ('-'). AP2D, etc. as in COSY. To
plot, use CPLB or CPPB. See Bruker manual for details.
Note:
Additional phase correction in the second dimension can be performed with the
'XF1P' command. The phase parameters can be established by reading a column
(RSC) and subsequent phase correction in EP. XF1P will then use those values.
7) The following plot parameters have been set by reading COSYDQB.500:
CX =CY =18; MAXY =24; DPO for X, Y-axis, offset=0.
Adding trim pulses at the end of an acquisistion might possibly be useful with other experiments as well.
AM-500: 7/25/91
Inverse probe
90deg. Reverse Mode Decoupler pulse @ 0H = 8.2 usec.
@ 1H = 17 usec.
5 mm Broad Band Probe:
90deg. Decoupler pulse @ 0H = usec
90deg. Decoupler pulse @ 0H = usec (reverse mode)
@ 1H = usec
@ 2H = usec
________________________________________
AM-400:
QNP Probe: (2/14/92)
90deg. Reverse mode Decoupler pulse @ 0H = 10.3
@ 1H = usec
@ 2H = usec
@ 6H = 37.5 usec
Inverse Broad Band Probe:
For the BB probe the
90deg. decoupler pulse @ 1H = is about 9 usec.
On the Console: Flip the NORMAL/REVERSE switch to REVERSE (up) .position
Fill out the Log book, please! If something was omitted, the next user might have a chance to figure out what to do!
Last Update: 12/23/94.