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ROESY
Set up appropriate 1H parameters. Normally should
acquire 1D 1H spectrum first.
pw = 1H pulse, should be 90º pulse
tpwr = high power for 1H; should be set according to
pw, or is correlated to pw.
nt = number of scans, should be multiple of 16, but 8
is acceptable
ni = number of t1 increments, points in t1 dimension,
at least 200, 256 is preferable
np = number of points in t2 dimension, must be even
number, 2048 is standard
slpw = spin lock pulse width, must be a 90º pulse with
a pulse width of ~30-35 ms
slpwr = power of spin lock pulse, cannot be more than
49 units, preferably ~43 units, must correlate to slpw
d1 = relaxation delay; normally should be longer than
in gCOSY to decrease t1 noise, and make ROEs between different types of
protons more equivalent, so 2.0-2.5 seconds is normal, but aromatic (or
vinyl or methyl) protons usually need 3.0-3.5 seconds for better spectra
mix = mixing time; the larger the mixing time the more
ROEs will be observed, but the less difference in intensity by distance. In
addition, the mixing time is correlated to the size of the molecule, small
molecule, long mixing time; generally 200-300 ms (so mix = .2 or mix = .3)
for MW ~400-2000 for longer mixing time 100-150 ms for short mixing time;
300-500 ms for long mixing time for MW < ~400
Steps:
1) Acquire 1 scan of 1H 1D experiment. Set cursors ~0.5
ppm beyond last proton resonance on both sides of spectrum, type command:
movesw
2) Re-acquire 1H 1D experiment (this is only required
for 1H 1D on side of 2D spectrum)
3) Move parameters to another experiment. To move
parameters from experiment 1 to experiment 3 type command:
mp(1,3)
4) Change to experiment 3, type command:
jexp3
5) Set ROESY parameters with setup macro, type
command:
ROESY
6) Spin will likely turn off, if it does not, open
Acqi window and manually turn it off. Lock Level should not drop more than
~5 %-10 %. If it does, you should reshim non-spin shims- X1, Y1, XZ, and
YZ, and on higher field (500/600) X2Y2 and XY
7) Set the number of scans (nt) to appropriate value,
generally 8 (it is somewhat preferable for nt to be a multiple of 16 but nt
= 8 is acceptable; nt might need to be increased for sufficient
signal-to-noise), type:
nt=8
8) Set the number of points in t1 dimension (second
dimension) (ni), generally 200-400 are suggested, the greater the points,
the better the resolution, and the less t1 noise observed, type:
ni=256
9) Set d1 time, normally should be longer than in
gCOSY to decrease t1 noise, and make ROEs between different types of
protons more equivalent, so 2.0-2.5 seconds is normal, but aromatic (or
vinyl or methyl) protons usually need 3.0-3.5 seconds for better spectra
10) Set the mixing time (parameter = mix); the larger
the mixing time the more ROEs will be observed, but the less difference in
intensity by distance. In addition, the mixing time is correlated to the
size of the molecule, small
molecule, long mixing time; generally 200-300 ms (so
mix = .2 or mix = .3) for MW ~400-2000 for longer mixing time 100-150 ms
for short mixing time; 300-500 ms for long mixing time for MW < ~400
11) Start acquisition, type command:
go
12) At end of experiment, set appropriate weighting
functions and linear prediction parameters, type commands:
setLP1
cosineroesy
13) If fn parameter now equals 4096, processing will
be slow, then type:
fn = np
fn1 = fn
14) Process 2D type command:
wft2da
15) Switch f1 and f2 axes (make f2 the x-axis), type
trace = 'f2'
16) Display as contour plot, type command:
dpn10
17) If spectrum has yellow streaks and purple streaks
horizontally across the spectrum, then the spectrum should be phased; set
cursor on peak, type:
ds
then manually phase, be careful to only click once on
spectrum during phasing, only rp (or rp1 phase correct should be changed)
redraw 2D type:
dpn10
If spectrum still has streaks in horizontal direction
more phasing is required including lp (or lp1) phase correct. If spectrum
has yellow/purple streaks in vertical direction, then the other dimension
needs to be phased, so switch the axes, type:
trace = 'f1' (or 'f2', depending upon which axis is
currently the x-axis, then type:
dpn10
18) Spectrum should be appropriately referenced
already, but you should confirm this; if necessary re-reference by putting
cursor on appropriate diagonal peak and type (assuming CDCl3):
rl(7.26p)
rl1(7.26p)
dp10
19) Adjust vertical scale with vs +20% and vs -20%
menu buttons or with middle mouse button or manually changing the
parameter, vs2d, so vs2d = 100 (the lower the number, the less noise
displayed), then redraw 2D with dp10 command
20) Print with 1D on side with 1D in experiment 1:
plcosy(10,1.2,1)
21) or Print with full rectangle, type:
full
dp10
pcon(10,1.2) page
22) Save data, type:
svf('filename')
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