196 (327) BRUKER BIOSPIN User Manual Version 002
PISEMA
Instead of raising the power level for
15
N, the power level for
1
H is reduced by
about 1.7 dB. Then the new 2
pulse in the tilted frame is:
In our example of a contact power level of 50 kHz on
15
N one would then calcu-
late for cnst17 = 40 807.0, giving an offset frequency of 28855 Hz for the LG fre-
quency, which is calculated automatically.
5. In order to verify all calculated power levels and offset frequencies, optimize
for the appropriate power level using the pulse program cplg.
6. Change to the desired PISEMA sample. Since a powder contains all possible
spin pair orientations, the measurement of an oriented sample is much recom
-
mended, because it is not only much faster, but also allows to judge the perfor-
mance much better. N-acetly valine can be rather easily grown to sizeable
single crystals and has all properties for a good setup sample: decent proton
T1, well defined sites and therefore narrow resonances. For a good crystal, the
residual proton line width will reflect the quality of the setup. Beware of crystal
twinning. The orientation of the sample should be selected carefully.
7. If the sample does tune and match very differently than the setup sample,
check the HH conditions briefly and verify that the found parameters are valid.
Correct power levels or pulse parameters if needed. This is especially impor
-
tant for saline lipid water mixtures.
8. Depending on the decision taken in (4), set
a. either PL13 = PL2 (case 4a) and set PL11 to the optimized value, higher pow-
er, i.e. a value of about 1.8 dB below pl1;
b. or set PL11 = PL1 (case 4b) and PL13 to the obtained value in the cplg experi-
ment, i.e. about 1.8 dB higher than PL2, which is less RF power.
9. Depending on the orientation of the single crystal, O1 and O2 need to be re-
optimized since the peak positions will change. Select an orientation that
shows the most low-field
15
N peak positions, because that will usually corre-
spond to the biggest
1
H-
15
N dipolar couplings.
10. Reoptimise power levels for the HH-contact.
11. Create a new experiment and setup a 2D data set, using the 1,2,3-icon. Load
the pisema pulse program. Go into the eda window.
12. Make sure the correct nucleus is selected in the F1 dimension.
13. In order to set the t
1
increment, go into the ased window clicking the pulse
symbol, and choose l3 to be 1, 2 or 3. This sets the t
1
increment and the pa-
rameter in0 is updated. Use the calculated value and set inf1 in the eda win-
dow correspondingly. Since dipolar couplings between
1
H and
15
N can be up
to 15 kHz, the spectral width (including the experiment scaling factor) should
be min. around 20 kHz.
1
_1
21
_12
67.0)(sin
−−
==
resonmreson
BB
LG
θτ
π
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