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NMR for Dummies
by Chad Rienstra
Reminders for All Experiments
==========================
1. Magic angle: KBr
a. Spin at 8 kHz for the 600 MHz spectrometer.
b. Use 1pulse pcode. DW = 5 usec. (Use previous parameter set from standards if
possible.)
c. Tune probe to 79Br (slightly lower freq. than 13C). If just "checking" the magicangle (unlikely it will need adjustment), it is sufficient to acquire a spectrum
without changing the probe tuning.
d. Setpw90X to ~3 us, aX90 = ~0.7
e. Acquire for 10 to 20 ms. Add ~100 Hz LB, FT, and phase.
f. Normalize intensity. Click the "big" up arrow three times. The first order
sidebands should be off the screen (>12.5% of the centerband) and the second
order sidebands should be close to the top of the screen (~10-12%).
2. Shimming: adamantane
a. Use 1pda_pm program.
b. If possible, start with a set of shims known to be "good" for the probe in use.
c. Spin ~5 to 8 kHz
d. Optimize TPPM condition: on resonance, aHdec = ~0.4, pw = ~11-12 us,
p2 = ~15
e. Set acquisition period length to 100 ms.
f. Autoshim orders 1 and 2. Or if you must do it the slow way, manually shim.
g. This should be sufficient if probe has been shimmed before.
h. An acceptable line shape for adamantane (600) is ~5 Hz FWHM, ~35
Hz at 3% (5 clicks).
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3. Decoupling: glycine
a. With cw decoupling, maximize intensity of CH2 signal by varying 1H carrier
freq (sf2)
b. Useamplitude aHdec ~0.6.
c. Optimize (as 2D array) pw and p2. Initial guess values: pw = 8 us, p2 = 15
degrees.
d. Always use the CH2 group as the calibration standard. It is the hardest to
decouple.
e. Fix p2 to the optimal value, and compare peak height over a range of aHdec and
pw. Suggested range: aHdec {0.4,0.7,0.05}, pw {7,12,0.3} us
Sample Changing
===============
1. To stop spinning with 3.2 mm module
a. Switch from auto to manual mode, at the same MAS rate from which auto mode
was started
b. Slowly decrease drive and bearing down to ~5 kHz MAS.
c. Rapidly decrease drive and bearing
2. Spin new sample up
a. In manual mode, bring bearing up to ~8-10 psi and then immediately follow with
drive to ~8 psi. A 3.2 mm rotor should spin up to ~8 to 10 kHz under these
conditions.
b. If tachometer reads >12 kHz and/or the signal is noisy, put signal on the scope.
c. If necessary, "remark" the black mark on the top spacer piece.
d. Repeat a. thru c. as necessary.
3. Lock spinning to desired rate
4. Check VT
5. Check probe tuning
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a. If known to be "far" off, use Network Analyzer to get within ~0.2 MHz.
b. Use short (~100 us), medium amplitude (~0.5) pulses such as the standard
tuneup program DCP (standards directory).
c. Compare forward to reflected voltage at the directional coupler. The ratio
should be greater than 10:1 for sure, and preferably greater than 20:1. If less
than 2:1, it will probably be necessary to return to step a.
1D 13C or 15N
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1. Use "vacpX_pm*" or "tancpX_pm*" pcode. These programs differ in the following
ways:
a. In vacpX_pm, the CP ramp on the X channel is defined by three parameters:
i. aXcp (beginning of ramp)
ii. steps (number of steps in ramp)
iii. step (size of step)
b. In tancpX_pm, the CP ramp on the X channel is defined by three parameters:
i. aXcp (MIDDLE of the ramp)
ii. deltaX (size of the ramp; total range is from aXcp - deltaX to aXcp + deltaX)
iii. betaX ("shape" of the ramp; see Meier papers for details. In brief, small
betaX results in "flat" ramp with fast wiggles at the end; large betaX results
in linear ramp over the range ±deltaX from aXcp; the best value to start with
c. WARNING: the number of steps in the tangent ramp is at present set to 512 and
is NOT user adjustable. This means that the ct time must be at least 100 us to
avoid crashes. (200 ns event time; there is a way to make this faster but I have
not done so in this code since the typical ct is about 500 us or longer). Of
course, like all things in pcode, this COULD be changed if so desired. However
I have made certain assumptions about use and left the program very UNuserfriendly mostly out
of laziness.
d. In my opinion, tancpX is easier to use (more intuitive) and it definitely gives
more options for optimization (in the limit of betaX >> deltaX, the shape is
linear, etc.)
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2. For the chosen pcode, optimize the CP parameters.
a. To start, fix aHcp near 0.6 and ct near 1 ms. As usual, it is easiest to
use a standard parameter set as the initial condition. You learn less this way
but get data faster... the ever-present dilemma of graduate school.
b. Fix the "shape" parameters (ii. and iii. from a. and b. above) to typicalvalues but
with a relatively small ramp (i.e., a ramp of total size 0.04 or less)
c. Map out the "finger spectrum" by varying aXcp. Note that with a very small
ramp, the fingers will be narrow, and with larger ramps, the fingers will
broaden.
d. After finding the best single condition, perform 2D optimizations of the ramp
size and shape. It is easiest to do so if the center of the ramp is fixed.
Now do you see why I like tancpX?
3. After CP is well optimized, return to decoupling (above) and compare performance
at various power levels for the different protonation states of 13C in your test sample.
For example, at which power level are the CO's sufficiently decoupled?
The CH's? The CH2's? Let's define "sufficiently decoupled" as the level at which the
signal would increase by less than 10% if the power is raised by 10%. Typically this is
somewhere around 0.6 to 0.7.
RFDR 2D C-C
===========
1. Start with H-C CP conditions from 13C 1D spectrum (e.g., vacpX_pm).
a. if using "va" 1D pcode (e.g., vacpX_pm), open RFDR_2Dc
b. if using "tan" 1D pcode, RFDR_2D_tan
c. otherwise change parameters step, steps, aXcp and betaX, deltaX.
d. See discussion above about difference between these parameters. You should
be able to "translate" from one set to another, and test to make sure you are
speaking the right language by the CP intensity.
2. Confirm signal and proper phasing.
3. Place 13C carrier freq (sf1) in middle of spectral region, ~90 ppm.
4. Optimize 90 degree pulses on 13C (aX90 and pw90X) for total signal.
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b Choose a method of optimization of mixing conditions.
a. "Fast" 2D's: choose bandwidth and resolution that allows a 2D
spectrum to be acquired in a minute or two (e.g., dw = 2 taur, al = 64,
na = 2, pd = 2, would be four minutes and give a decent (but folded)
NAV or Tyr 2D). In this case, you would watch for polarization transfer
as buildup of cross peaks. This is the foolproof way.
b. 1D's where the polarization starts primarily on one set of spins, e.g., the directly
protonated ones. This can be accomplished by setting the CP period to be ~100
us. See above regarding the tancpX warning if you are using RFDR_2D_tan. I
will fix this sometime. In any event, in this case you will observe polarization
transfer as a buildup of the CO peak. This is the sometimes tricky way, but is
faster. (Another graduate student dilemma here.)
c. Perhaps you could start with b., and then once you think things are working,
return to a. to confirm.
6. Observe polarization transfer as a function of the mixing parameters:
a. RAD: aHrad, tau_rad
b. RFDR: q (number of pi pulses), pi pulse length (always set pw180X and aXzq
to be a sensible combination)
7. Choose bandwidth and digital resolution in the t1 dimension.
a. Preferably set dw1 so that taur is evenly divisible by dw1 (e.g., at 10 kHz MAS,
taur = 100 us, dw1 = 12.5 us)
b. Under "normal" circumstances (such as a standard broadband 2D), the desired
bandwidth is >200 ppm.
c. The maximum evolution time in t1 is limited by sample heating primarily.
Consider the direct dimension acquisition time and the maximum evolution time
in t1, in terms of your sample's salt content, thermal stability, etc.
d. WARNING: the memory board depth on the D-board is 32 MB. This means
that the total number of complex points acquired must be less than 4 M.
For example, al =2048 and al2 = 2048, or al = 4096 and al2 = 1024.
8. Acquire the real 2D. Well, also the imaginary part. I guess this should
say acquire the actual 2D.