Decoupling


dmf (or dmf2)

= 1/pw90 (Varian specification)
Then type dmfadj (or dmf2adj) to properly set dmf (or dmf2)

Waltz decoupling

bandwidth = ±1.1 • 1/(4 • pw90) - cleaner than garp (dres set to 90 for Waltz - you can check this by looking at the WALTZ16.DEC file). Waltz16 is good for N15 decoupling.

Garp

bandwidth = ± 2.2 • (1/4 • pw90) - do not use in the middle of a pulse sequence since it commonly creates decoupling side band artifacts in multi-dimensional spectra. Set dres2 = 1 (check GARP1.DEC file). Garp gives sidebands in N15 decoupling but is good for C13 decoupling.


13C Decoupling power calibration:

Kay sequences
  • se hnco_ydw_cal and set spcx = 'hard', d_cx = 44, array pwco90x (on the 800 we obtain a pw90 around 80 usec at this power).
  • The field strength = 1/(4 • pwco90x) (in this case 1/(4 • 0.00008) = 3125 Hz
  • If this is used in GARP for C13 decoupling the bandwidth would be ±2.2 • 3125 Hz. (But remember to set dmf = 1/pw90 and type dmfadf
BioPack
  • on 500 want about +/-60 ppm range. If using garp, 60*125/2.2 = 1/(4*pw90) give pw90=73us. Using gChsqc with aromatic region gives pw90=72 at 45 power.
  • on 800 can decrease bandwidth to avoid high powers to +/-45 ppm. This gives 62us at 45 power.

15N Decoupling power calibration

  • A good 15N decoupling power = 44.
  • For N15 decoupling from 102 to 132 ppm. On 500 we have 30x50.6=1520 Hz, so ±1000 Hz should be plenty. pwN90=1/(4 • 1000)=250.
  • On 500 we have Npower=40 with pwN90=245 giving dmf2=4082.
  • On 800 we are using decoupling power of 42 with dmf2=4828 giving pwN90=207 (±1200 Hz) with Kay's sequences. For BioPack we have +/-1500 Hz with 165us at power=46.
  • For BioPack use ghn_co to calibrate pwN for decoupling.

Carbon decoupling in HSQC and 15N relaxation experiments

  • c_flg on HSQC and N15 experiments uses a 180° composite pulse (composed of 4 90° pulses) on 13C
  • On the 800 the composite pulse does not work well. Therefore, we need to use an adiabatic inversion pulse. Run apod_chirp to set up an adiabatic WURST pulse (see HNHA-jr experiment). Typically use a 400 usec 80 kHz bandwidth pulse. Should be able to use pulse tool to view this

Deuterium Decoupling

First, read the instructions SetupTab -> CalibrationsPage -> 2H DecouplingInstructions button

Note: channel 4 has 10 dB attenuation on amplifier at the time this was written.
There are 2 methods in BioPack:
(1) direct 2H observe
this will automatically put calibrations in probe file, but you must recable
(2) 13C observe using C-D coupling
you can actually see the effect of decoupling, but you have to manually edit probe file
S.E. recommends method #1 and then check using 2H dec test with 13C observed on deuterated benzene

Direct 2H observe using Channel 4

Use D2O/H2O sample; either the D2O sample used for lock when not running exp, or your sample
recable relay on magnet leg as described in instructions
SetupTab -> CalibrationsPage -> 2H Decoupling 4th Channel
Will find power for 90deg with pw of 200 us, such as 192 us at power=59
Dec will use pw of 500 us, so pwr scaled by macro. After calibration, HCN probe file should have about 500 us pw90 (dmf=2000) at power=52 and dof=-350
put cables on magnet leg back the way they were

Test using deuterated protein

high resolution ghn_ca with sw1=3000, ni=512, and C-beta decoupling (Cbdec) off. Broad peaks (in C dimension) without 2H decoupling, narrow doublets with 2H decoupling (cwc).

Calibrate using 13C observe

This method allows you to actually see the effect of decoupling, but you have to manually edit probe file. The directions below were written with previous Vnmr versions so you will have to modify for latest vnmrj software.
use deuterated benzene sample
-> Setup -> Proteins -> Calibrate -> Other -> Deuterium -> 13C observe -> 2H pw90
Do not change cables - should already be setup for 13C observe
array('pwx3',20,0,50)
tpwr=61, pw=13 (or whatever 13C should be)
d1=5, sw=3000, fn=8k, vp=40
dpwr3=52 (or whatever you think dec power should be to give 500 us pw90 for channel 4
ga to collect spectra
phase so center peak is positive and find null to give 2H pw90. Adjust power and repeat to find values for 500 us pw90. Should be same as method #1
pw90 is not written to probe file. You will have to manually edit ~/vnmrsys/probes/HCN/HCN. dmf is calculated from pw90 (=1000000/pw90) and should 2000 for 500 us pulse
find dof3 using sequence below

Test decoupling and find dof using deuterated benzene

-> Setup -> Proteins -> Calibrate -> Other -> Deuterium -> 13C observe -> 2H dec test
ignore instructions to set dmm3 and dm3! You want to use the default values of dmm3='ccw' and dm3='nny'
tpwr=61, pw=13 (or whatever 13C should be)
d1=4, sw=3000, fn=8k vp=40
dpwr3=52 (or what was found for 500 us pw90 above) and dmf3=2000 (=100000/pw90)
should see only one peak. Test by arraying dpwr3=0,30,40,52 (or whatever) and you should see triplets disappear at higher dec powers
find dof3 by array('dof3',9,-2000,500)
dof3 for HCN probe file is given by dof3=dof3-4*dof3.
I found dof3=-900 with this method, compared to -350 for method #1.