NMR Spectroscopy Short Course 2007 |
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Experimental
Guide for INOVA-400 NMR
Spectrometer Introduction: NMR Experiment Procedures Experiment 1: 1D proton and Basic Operations Experiment 2: 1D Carbon and Basic Operations Experiment 3: Proton 90° Pulse Width Calibration Experiment 4: Carbon 90° Pulse Width Calibration Experiment 5: APT (Attached Proton Test) Experiment 6: DEPT Experiment 7: INEPT Experiment 8: Presaturation Experiment 9: COSY Experiment 10: HETCOR Experiment 11: 1D NOE Experiment 12: 1D Carbon with Complete Fluorine Decoupling by Using Wave Function Generator Experiment 13: Variable Temperature Experiment
Introduction: NMR Experiment Procedures Step 1. Login the Computer System All modern NMR spectrometers are controlled by host computers. For the security and administration reason, all research groups using NMR in Emory University are issued their group usernames and passwords for the host computers. All authorized NMR users need to use your own group usernames and passwords to logon to the host computers. Step 2. Change Sample A standard CDCl3 or other deuterated sample is always placed in the magnet to keep the instrument in working condition when nobody is using it. Every user needs to replace the standard sample with your own sample at the beginning of each experiment and change back at the end of experiment. Step 3. Lock and Shim the MagnetLocking the magnet keeps its field constant and stable
during data acquisition. Shimming the magnet makes it uniform and homogenous for
a sample. Step 4. Setup Parameters and acquire DataRetrieve
default parameters that are setup and updated for you by the NMR Center so that
you may collect your data efficiently. Step 5. Process Data and Plot SpectrumThis
step converts data into visible NMR spectra. Step 6. Logout the Computer SystemYour
mission is completed and you need to do a few things for next user. The
step 1, 2, 3, and 6 are the same for all experiments. We will describe them in
detail in Experiment One. The step 4 and 5 differ from experiment to experiment. Experiment One: 1D Proton and Basic Operations Sample
to be used: 10% Strychnine in CDCl3 Step 1.
Login the Computer System
1.1
Type in your username at login prompt-------- INOVA400> username Type
in your account password------PASSWD> password
The VNMR software will be loaded in several seconds with the display of four windows. The upper left window is used for entering commands and selecting menu buttons; the middle left for displaying NMR spectra and the lower left for displaying NMR parameters and other text messages. The upper right window is called “ACQUISITION STATUS” window, indicating the working status of the instrument, such as lock level, spin rate, temperature and acquisition time, etc. Step 2. Change Sample2.1
2.1.
Click on the Acqi or type acqi in the command line to
display the fifth window at lower right, which is called “ACQUISITION” window. All
the operations hereafter in Step 2 and
Step 3 will be operated. There are CLOSE, LOCK, FID, SHIM and LARGE popup menu
buttons, and Sample eject and Sample insert buttons. We will be focusing on the LOCK and the SHIM buttons first. 2.2
Click on LOCK button to display lock sub-window then click
SPIN: off button to turn off sample spinning; click
LOCK: off to turn off the lock. 2.3 After a few seconds, click on the
SAMPLE: eject button to eject the standard sample onto the
top of the magnet; remove the spinner, replace the sample with your own, use
sample depth gauge to measure the correct tube position, put the spinner back
onto the top of the magnet again. Caution: place the spinner with a sample on
top of the magnet only when ejecting air is on. 2.4 Click on the SAMPLE: insert button to
put the spinner back down inside the probe. Wait for 3 to 5 seconds, then click
on the SPIN: on button to make the sample spin. In this step, you need to adjust lock and shim parameters by using the mouse. Each parameter can be increased or decreased by 1, 4, 16 and 64 units as shown by corresponding –1+, -4+, -16+ and –64+ buttons. For example, when you place the cursor on a button, say –4+, if you click the left mouse button once, the corresponding parameter is decreased by 4 units; if you click the right mouse button twice, the parameter is increased by 8 units. 3.1
If you are using CDCl3 solvent, the magnet could be locked almost
instantly most of the time. This is indicated by the display of lock signal
plateau and a green LOCKED message below it. Then you directly go to step
3.3. 3.2
If you don’t see a plateau signal with a LOCKED message or you
are using solvent other than CDCl3, click on LOCK: off button, adjust
Z0 to make wave-like lock signal into plateau-like resonance, then click LOCK: on button. A green LOCKED should be shown. Then
adjust the lock phase to make the lock level as high as possible. You need to
reduce the lock power to 26-28 and lock gain 30-36 if the lock
level excesses 100. You may also
check our record sheets on the desk. There are Z0, Lock power, Lock gain, and
Lock phase values for different solvents recorded by previous users. 3.3 Now it is shimming time. There
are as many ways of shimming as NMR users. We suggest you shim it this way:
3.2.1
Click SHIM button to display shim sub-window. The lock
level is shown in this window as two color bars and exact lock value is shown
underneath the bars. In the middle of the window, there is one triangular button
after ‘SHIM:’ which let you switch from shim sub-windows and lock
sub-window by just click on it. Now it should show coarse z by the
button. 3.2.2
Adjust Z1C (Z1 coarse) and Z2C by alternatively changing 1 or 4 units to
increase the lock level as high as you can. If the lock level increases to 100,
decrease lock gain and then continue to adjust Z1C and Z2C. Higher lock level
indicates better homogeneity. 3.2.3
Click on triangular button to switch to fine z sub-window. Adjust
Z1, Z2 and Z3 to make the lock level as high as possible.
We do not suggest you to adjust Z4 and Z5 at this stage. If you really
want to adjust them, it is better write them down before you change them, so
that you are able to go back in the case you get a worse shimming. 3.3
Finally click the CLOSE button to end the shimming
procedure.
Tip 1. Reinstall our default
standard shimming file.
If
you have a very bad shim, type “loadshim” in the command window to
reinstall our default shim file for the standard sample. Step
4. Setup Parameters And Run 1D proton Experiment 4.1 Type h1
to load the standard parameter for proton with a default CDCl3 solvent. If your
solvent is not CDCl3, you may simply type h1(‘solvent’), where the solvent may be one among acetone,
DMSO, D2O, etc. You may also type h1 and then solvent=’DMSO’
etc.
Proton standard parameters.
4.2 Type nt=16
or 32, according to sample concentration. 4.3
Type ga
(submit to acquisition) to begin
acquisition. 4.4
After acquisition is completed, a proton spectrum should be displayed on
the screen now. If not, type wft (Weight and Fourier transform) to show it. Then enter aph
(automatic phasing) to phase the spectrum. Type vsadj
(vertical scale adjustment) to adjust the vertical scale of the spectrum
automatically. Type dscale or click on Dscale button to display chemical shift scale
under the spectrum. Tip 2.
Manual Phasing If
you need more improvement on the phase, you may manually phase the spectrum by
clicking on Phase button first and then clicking on the region of
interest and moving up or down the mouse while pressing LMB to adjust its phase.
You may continue to click the cursor on the next region and adjust the phase
same way. Clicking on the Box or Cursor button will exit the
manual phase routine.
Tip 3.
Manual Vertical Scale Adjusting Place the cursor
above the base line and click middle mouse button to increase the vertical
scale. Place the cursor below the base line and click middle mouse button to
decrease the vertical scale. Step
5. Data Processing
5.1
Zoom operation Click
LMB on the left side of a peak and RMB on the right side, two vertical red
cursors appear at the click points. Then click the expend button on
the menu bar, the region between the red cursors will be expanded. Just click on
full button to get back to the full spectrum
after you did expending, or type f then press enter. You may do this
zooming operation as many times as you like. 5.2 Set reference After zooming the region containing the reference
peak, click the LMB around the top of the peak, type nl to place the cursor on
the top of the peak exactly. Then type rl(x.xxp)
to set its chemical shift. For example, rl(4.80p) will set a peak frequency to 4.80ppm. 5.3 Integral operation
Enter vp=20. Type ds
cz
to clear all the previous integral values in the buffer. Click on Part Integral
button for the full spectrum or its expanded region to display an integral
curve. Then click on Resets button. Now click LMB at
both sides of each peak or integral region to get separated integral lines of
the spectrum. If
you know the proton number a specific peak presents, you may set the value for
that integral as follows: placing
the cursor on that integral and click on Set Int. The computer will then
ask what value to assign that integral. After you put the value for that
integral, the computer will recalculate the value of every other integral. You
are able to see these values by typing dpir.
Tip 3.
Redo integral operation If you made a mistake during the integral operation,
You can redo the integral operation by click on Full Integral and
then No Integral button to clear the integral line.
Then type cz, click on Part Integral button
and repeat the procedure 5.3. 5.4 Peak picking operation Type ds
and click on Th buttons to display a yellow horizontal threshold
line. Press and hold LMB to move the yellow cursor to the position you desire,
then click on the Th button again. Type command dpf
to display peak frequencies above spectrum peaks. Tip 4. Back to interactive
adjustment
During above operations, if you are not able to find the button you need or you are not be able to use red cursor, just click on Main manue > Display > Interact
5.5 Label your experiment data Type ctext
to clear any text label for the present experimental data.
Type text(‘Your
sample name, date\\solvent, e’) to label the present experimental
data. Command dtext will display
the text on the top left corner of your spectrum. 5.6 Print
and plot spectrum and related information pl---to
begin print the present spectrum; pap---to print all the parameters including the text
labels, if any. ptext---to print text labels without other parameters. pscale---to print the spectrum scale in ppm or hz by axis=“p”
or axis=“h”.
ppf---to
print all the peak frequencies over the respective peaks.
pll---to print a list of the frequencies and intensities of
all the peaks in the spectrum.
pirn---to
print normalized integral values below the respective peaks.
pir---to
print proton numbers below the respective peaks.
page
---to expel the paper from printer. Remember, every printing procedure starts with pl
and ends with page.
You can put other printing commands selectively between these two commands. 5.7 Save your experimental data and
reload your experimental data
You may save your spectrum data. The
directory path is /export/home/nmrusr/Login_ID/ when you login. It is your group
directory. You should make a directory of your own files. Type
mkdir(‘yourname’) to make a directory of your own. The
computer automatically goes into your directory /export/home/nmrusr/Login_ID/yourname/.
If it does not, click on Main manue > File then highlight
your directory name and click on Set director.
Remember you can go back to your group directory at any time by typing cd. Type
svf(’file_name’) to
save your data in the form of FID. 5.8.
To reload your file, go to your directory first as described above then
click on Main manue > File >
highlight your file > click on load. Step
6. Logout the Computer System 6.1 Retrieve standard file and
replace your sample with the standard.
Type h1
to set up standard proton file. This is especially important after you
have done a carbon-13 experiment. It will turn off the decoupling channel
automatically.
Then see Step 2 for details about replacing NMR sample tubes. 6.2 Lock and shim the magnet See Step 3
for details. You are required to shim the magnet to the required level: lockpower=32,
lockgain=40 with lock level of over 75%!!! 6.3 Exit the VNMR program Type exit
to close the VNMR program. Now you will see a full screen of Varian’s logo. 6.4 Exit UNIX system Press the RMB to select Exit item in the popped up menu, release RMB. You will be prompted to select one of two choices: Exit or Cancel. Click on Exit to exit UNIX system. The screen will become blank and a prompt appears. 6.5 Logout
Type logout. Sign off the
logbook for the instrument. Experiment Two: 1D
Carbon-13 and Basic Operations
Sample
to be used: 30% Strychnine in CDCl3 Step 1. Login The Computer System(See
Experiment One) Step 2. Change Sample
(See Experiment One)
(See Experiment One) Step
4. Setup Parameters Carbon Experiment 4.1
Standard C-13 spectrum (Fully decoupled with NOE enhancement):
Type c13 to call up the
standard carbon-13 experiment parameters with CDCl3 solvent.
4.2
Enter nt=256, according to the
nature of your sample. 4.3
Type ga
to begin acquisition. 4.5
After
acquisition is done, a carbon spectrum is displayed. If not, enter wft.
Type aph
for automatic phasing. Manual phasing is often necessary for a carbon-13
spectrum. Step
5. Data Processing
5.1 Zoom operation
Click LMB on the left side of a peak and RMB on the right side, two
vertical red cursors appear at
the click points. Then click the Expand button on the menu bar, the region
between the red cursors will be expanded. You may do this zooming operation as
many times as you like. 5.2 Set
reference After zooming the region containing the reference peak, click the LMB
around the top of the peak, type nl
to place the cursor on the top of the peak exactly. Then type rl(x.xxp)
to set its chemical shift. For
example, rl(77p) will set a peak
frequency to 77.0ppm. 5.3 Peak picking operation Type ds
and click next and th
buttons to display an horizontal threshold line. Press and hold LMB to move the
yellow cursor to the position you desire, then click on the th
button again. Type command dpf
to display peak frequencies above spectrum peaks.
5.4 Label your experiment data
Type ctext to clear any text
label for the present experimental data.
Type text(“Your sample name,
date\\solvent, etc”) to label the present experimental data.
Type dtext to display the text
label of present data on the upper left of the spectrum.
5.5 Print
and plot spectrum and related information pl----to
begin print the present spectrum;
pap---to
print all the parameters including the text labels, if any. pscale---to print the spectrum scale in the unit of ppm or hz by axis=“p” or
axis=“h”.
pltext---
to print the label. ppf---to print all the peak frequencies over the
respective peaks.
page
---to expel the paper from printer. 5.6 Save your experiment data
You have to save your data in /export/home/nmrusr/Login_ID/yourname/
Type svf(’file_name’) to
save your data in the form of FID. Step 6. Logout the Computer System6.1 Retrieve standard file and
replace your sample with the standard.
Type h1 to set up standard
proton file. That will turn the decoupler power off too.
Then see Step 2 for details about replacing NMR sample tubes. 6.2 Lock and shim the magnet See Step 3
for details. Before clicking Disconnect, you are required to shim the magnet to
the required level: lockpower=32,
lockgain=40 with lock level of over 75%!!! 6.3 Exit the VNMR program Type exit
to close the VNMR program. Now you will see a full screen of Varian’s logo. 6.4 Exit UNIX system Press the RMB to select Exit item in the popped up menu, release RMB. You will be prompted to select one of two choices: Exit or Cancel. Click on Exit to exit UNIX system. The screen will become blank and a prompt appears. 6.5 Logout
Type logout. Sign off the
logbook for the instrument. Experiment Three: Proton 90° Pulse Width CalibrationSample
to be used: 10% Strychnine in CDCl3 Step 1. Login The Computer System(See
Experiment One) Step 2. Change Sample
(See Experiment One)
(See Experiment One) Step 4. Setup
Parameters and Run Experiment 4.1
Type
h1 to retrieve standard proton experiment parameters. 4.2
Set d1=1
nt=1
4.3
Type ga
to begin acquisition. 4.4
Expend the spectrum between 1ppm away from the left of the left most peak
of the spectrum and 1ppm away from the right of the right most peak. Type
movesw to reduce the spectrum width and increase the digital
resolution. 4.5
Set d1=20
or a value which enables protons relax back to thermal equilibrium status. tpwr=58 4.6
Type array and input the following parameter and numbers as
computer requests: parameter
to be arrayed: pw. enter
number of steps in array:
20 enter
starting value: 2 enter
array increment: 2 You
are able to see the whole array parameters in the Text Window on the bottom by
typing da
4.7
Type ai
ga to begin acquisition. The ai means absolute intensity
mode. Step 5. Data
Processing
5.1
Display arrayed spectrum When
the acquisition is finished, enter ds(1) for displaying the first spectrum of the array. Type aph
for autophasing. Chose a singlet or a doublet peak to expend and then enter dssa
for displaying peaks at the same chemical shift range in all of the individual
spectrum horizontally. 5.2
Null point spectrum and 90° pulse width From
the arrayed spectrum, find out first null point (the peak intensity is 0), check
the pw value corresponding to it. The 90°
pulse width is this pw value divided by 2 at given power (tpwr). In this case
tpwr=58.
Type pw90=xx
to set up this parameter, where xx was obtained as above. 5.3
Label your experiment data
See Experiment One. 5.4
Print and
plot spectrum and related information Type
plww pap page to print spectra in
white wash mode (after the first spectrum, each spectrum is blanked out in
regions in which it is behind an earlier spectrum). 5.6
Save your experimental data
See Experiment One
Step 6.
Exit And Logout
See Experiment One Congratulations!!! You have finished a complete Proton 90° Pulse Width Calibration experiment.
Experiment
Four: 90° Pulse Width
Calibration for Carbon Channel Sample
to be used: 40% p-dioxane in benzene-d6 (ASTM) Step 1. Login The Computer System
See Experiment One
See Experiment One
See Experiment One 4.1
Type
c13(‘benzene’) to load standard carbon parameter set. Check
following parameters: tpwr=60,
nt=1, pw=6 gain=‘46’, then enter ga
to begin acquisition. 4.2 After the acquisition is finished, phase the spectrum if necessary. 4.3
Set d1=20
or a value which enables protons relax back to thermal equilibrium status.
4.4
Type array and input the following parameter and numbers as
computer requests: parameter
to be arrayed: pw. enter
number of steps in array:
20 enter
starting value: 2 enter
array increment: 2 You
are able to see the whole array parameters in the Text Window on the bottom by
typing da
4.5
Type ai
ga to begin acquisition. The ai means absolute intensity
mode. Step 5. Data
Processing
5.1
Display arrayed spectrum When
the acquisition is finished, enter ds(1) for displaying the first spectrum of the array. Type aph
for autophasing. Chose a singlet or a doublet peak to expend and then enter dssa
for displaying peaks at the same chemical shift range in all of the individual
spectrum horizontally. 5.2
Null point spectrum and 90° pulse width From
the arrayed spectrum, find out first null point (the peak intensity is 0), check
the pw value corresponding to it. The 90°
pulse width is this pw value divided by 2 at given power (tpwr). In this case,
tpwr=60.
Type pw90=xx
to set up this parameter, where xx was obtained as above. 5.3
Label your experiment data
See Experiment One.
See Experiment Three. 5.5
Save your experiment data See Experiment One. Step 6.
Exit And Logout
See Experiment One.
Experiment Five: APT
Sample
to be used: 30% 2-butanol in CDCl3 Step 1. Login The Computer System
See Experiment One.
See Experiment One. Step
3. Lock And Shim The Magnet
See Experiment One. Step
4. Setup Parameters and Run Experiment 4.1 type
c13 to load standard proton experiment parameters. 4.2
Set
nt=8. Type ga to begin acquisition
for a common 1D-carbon spectrum. 4.3 After acquisition, type aph
for automatic phasing and /or manual phasing if needed. 4.4
Type apt
to load standard APT parameters. Double check following parameters:
pw-----a
normal observe pulse. It need not be 90°
pulse.
p1-----180° observe pulse width.
d2-----the
tau delay time. d2=0.007 (7ms)will make CH, CH3 down and C, CH2 up.
d1-----the
pulse delay. It is set to around 10 seconds.
dm----decouple
mode. It is set as
dm=‘yny’ for APT.
nt------scan
numbers. It may be set to 32 4.5 Type ga to acquire data. Step
5. Data Processing
When
the acquisition is finished, type aptaph
macro to phase APT automatically. Type
pl
pscale pap page to plot the spectrum. Step
6. Exit And Logout Congratulations!!!
You have finished a complete APT experiment. Experiment Six. DEPT Sample to be used: 30% 2-butanol in CDCl3 Step 1. Login The Computer SystemSee Experiment One See Experiment One See Experiment One Step
4. Setup Parameters And Run Experiment 4.1
Type command c13 to setup standard
parameters for 1D carbon spectrum. 4.2
Set nt=4 or 8 to acquire an
1D carbon spectrum, and set the chemical shift. 4.3
Type dept to setup standard
parameters for DEPT experiment. 4.4
Check and/or change the following important parameters:
ss=4
steady-state pulse or dummy scans
pw=15
90° pulse for carbon-13;
tpwr=60
transmitter power of observer channel;
pp=14.6
proton 90° pulse from decoupler channel;
pplvl=60
proton pulse power from decoupler channel;
d1=5
relaxation time
j=140
average C-H coupling constant;
mult=0.5,1,1,1.5
arrayed pulses for CHx, CH, CH2 and CH3;
nt=32
number of scans, A multiple of 16 is suggested. 4.5
Type command ga to acquire data. Step
5. Data Processing 5.1
After the acquisition, type
ds(1) to display the first spectrum. 5.2
Phase the spectrum and select a proper threshold by using menu button of th. 5.3
Enter adept dssa to analyze and
display arrayed spectra of CHx, CH, CH2 and CH3. 5.4
Type pldept
to plot the DEPT spectra as displayed by dssa. Step
6. Exit And Logout
See Experiment One Congratulations!!!
You have finished a complete DEPT experiment. Experiment Seven. INEPTSample
to be used: 30% 2-butanol in CDCl3 Step 1. Login The Computer System See
Experiment One
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