Bruker BioSpin Solid State NMR, User Manual

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264 (327) BRUKER BIOSPIN User Manual Version 002
Double-CP
The setup for DCP can be rather much sped up and simplified by a python pro-
gram named dcpset.py. This program will ask for the 90° pulse widths and associ-
ated power levels and for the spin rate and calculate the appropriate power levels
for the HH condition for all pulses except for the proton channel.
Contact solids@bruker.de to receive this python program and some instructions
for use. This program serves as an example on how the experiment setup can be
controlled by the high level script language.
Setup of the 2D Double CP Experiment 20.2.4
1. Load a suitable sample, spin it up, set the desired temperature and match and
tune the probe. As a simple setup sample, full
13
C,
15
N-histidine may be used
(
d1
=10s, 2-4 scans,
p15
=1msec,
p16
=3msec).A labeled oligopeptide or small
protein will of course provide a more interesting spectrum. With proteins, good
results should only be expected if the preparation is micro-crystalline. In such a
case, water, salt and cryo-protectant (glycol, glycerol) will very likely be pres-
ent. This means that the probe proton channel will be detuned to lower fre-
quency, and tuning may be difficult, if not impossible at high proton frequencies
and salt contents. In such cases, E
free
probes are recommended.
2. Run standard 1D cp
13
C and
15
N experiments; determine the required offsets
for all frequencies and the required sampling windows.
3. Re-optimize the H-N and N-C HH conditions.
4. Generate a new data set and switch to 2D data mode, using the “123”-icon in
eda
.
5. In
eda
, set the pulse program to
doubcp
. Set FnMode as desired, usually
STATES-TPPI.
6. Make sure the correct nucleus (
15
N) is selected in the F1 dimension.
7. Set the sampling windows for both dimensions from the previously acquired
1D spectra.
8. Both acquisition times in F2 and F1 should be considered with care, since the
decoupler is on at high power during both periods. Especially for biological
samples, where the RF heating may be high and the samples are temperature
sensitive, it is essential not to use overly long acquisition times and high duty
cycles. Remember that the heating effect is generated inside the sample
where the temperature increases within milliseconds, whereas cooling re-
quires transfer of the energy to the outside of the spinner, which takes sec-
onds! E
free
probes eliminate these problems to a large extent.
9. The basic double-CP experiment can be extended into many different varia-
tions. One example is the double transfer N-C
α
-C
β
, where the second transfer
step is made selective so only α -carbons are polarized from the nitrogen, then
magnetization is transferred from the α -carbons to the adjacent β -carbons.
This can be done by a simple PDSD or DARR proton spin diffusion step, or by
a
13
C-
13
C homonuclear recoupling step (HORROR, DREAM, or other). Like-
wise, the N-C
α
-C
x
experiment transfers from the α -carbons to all (X) carbons
which are in close enough proximity to the α -carbons. Check with your applica-
tions support for appropriate pulse programs.