CHEM4629
Fall 2013
NMR Experiments and Instructions for Operating the Varian 400 MHz NMR Spectrometer
The Departmental NMR Instrument
The Varian 400 MHz NMR instrument that is used in CHEM 4629 is shared by undergraduate
students, graduate students and staff of the Department of Chemistry and Chemical Biology.
Please make sure you are confident in the operation of the instrument as repairs will require
considerable time. Ask for help if you need it and please follow the rules developed for timesharing on the NMR. Students will be provided the account name and password for the course
as well as the code for the lock on the door (press the 2 and 4 buttons on the lock together,
then 3).
You will be able to schedule time online for use during the week. The online scheduling site is:
http://faces.ccrc.uga.edu/login0.php4
The following are an abbreviated set of instructions for obtaining routine 1H and
13C
NMR
spectra on standard samples. As these instructions are brief and under constant revision
please do not rely on these exclusively. It is mandatory to participate in a “hands-on” training
session before using the spectrometer independently.
Log in
When you first view the screen on the NMR console there should be a login box
Enter the USERNAME chem4629 <return>
Enter the password pass4629 <return>
Click on the spectrum icon at the bottom of the screen.
The next screen consists of a black graphic (spectrum) underneath a series of menu “buttons”.
Below the black spectrum is a display region for the sample parameters. The “command” entry
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line is above the menu buttons. The top right hand corner of the screen contains a box
summarizing the status of the spectrometer.
INSERTING THE SAMPLE, LOCKING, AND SHIMMING MAGNETIC FIELD
Inserting the Sample
• Open the air valves on the wall to the left of the console and air tank if needed.
• Click on the button labeled “acqi”. A new screen will appear to the right hand side.
• Insert your sample into the plastic spinner, aligning the depth of the tube with the bottom of
the brass tube guide.
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• Click on “eject”. You should hear a whoosh of air.
• Place the spinner/tube assembly in the bore tube. It should float on the cushion of air. Be
sure it is floating before letting go. (Caution: do not insert sample if you don’t hear the flow of
air!!)
• Click on “insert”. The sample will be lowered into the probe. Listen for the change in pitch
and a click as the sample settles into the probe.
Locking
Under computer control, the lock system maintains the frequency stability of the spectrometer
as the static magnetic field generated by the superconducting magnet drifts slowly with time or
changes due to external interference. Locking makes the resonance field of the deuterium in
the deutered solvent coincide with the lock frequency.
• Click on “lock” on the screen
• Turn the spinner on by dragging the slide at the bottom of the screen to 20 Hz
click “spin: on” at top of screen if necessary.
• Find the Z0 value appropriate for your solvent, it will be listed on the cover of the NMR black
binder. The Z0 value for CDCl3 is typically about -260 dB. For any functions in this screen, you
can change the parameters by either dragging the slide with the mouse or by clicking the left
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(reduces numeric value) and right (increases numeric values) mouse buttons when the cursor is
on the appropriate screen buttons.
• Make sure the lock is “off”. Increase lock power to 40 dB and lock gain to max (64 dB). Move
Z0 until the sinusoidal function changes to a step function (see below).
•Once you have obtained the step function, turn the lock on by clicking the Lock:ON button.
The red message “not locked” will change to green “locked” and the signal will hit the top of
the screen. Reduce lockpower to 28 dB (if using CDCl3) then adjust the lockgain to bring the
signal to ~ 50% of full scale. It is best to operate at the lowest lock power and lock gain possible
while maintaining locked condition. This avoids oversaturation of the nuclei. Set lockphase to
that in the binder recommended for your solvent. The optima lockphase for CDCl 3 is currently
2180.
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SHIMMING
Shim coils are small magnetic fields used to cancel our errors in the static magnetic fields. In
shimming the current to the shim coils is adjusted to make the magnetic fields as homogenous
as possible. Computer-controlled digital-to-analog concerters (DACs) regulate the roomtemperature shim coil currents. Every time a new sample is introduced into the magnet or the
probe is changed, it is necessary to readjust the shims.
• Click on the “SHIM” button.
 To recall the best shim set parameters type rts(‘best’) su <return> in the input window.
Return to the SHIM window by clicking acqi
• To shim the magnetic field to get sharp signals, repeatedly adjust Z1C and Z2C with the
mouse (left hand button to decrease, right hand button to increase) to maximize the lock level
shown on the “thermometer” coarse display bar. (Shimming creates a more uniform magnetic
field around the sample resulting in narrower spectral lines.) The idea is to maximize the signal.
Adjust Z1C by mouse clicking the button until the lock level is maximized. Next, adjust Z2C to
maximize the lock level. As these two shims are coupled, iterate back and forth between Z1C
and Z2C until you can no longer increase the lock level. If the lock level goes off the top of the
coarse lock display bar (i.e. all red), decrease the lock gain until the lock level is ~50%, start
shimming again. Shimming is very important and will affect the appearance of your spectrum.
It is an acquired skill, so be patient and be ready to practice.
Improper shimming WILL broaden lines making calculation of coupling constants difficult or
impossible and produce out-of-phase lines resulting in incorrect integration values. In other
words – improper shimming will NOT allow you to correctly interpret the spectra
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• Start with the ± 16 buttons on Z1C and Z2C and do 2-4 clicks on each, switching between each
in turns, until any changes do not further increase lock level. Then repeat with the ± 4 buttons.
Do NOT adjust Z1 and Z2 on the Varian 400 MHz NMR. It is redundant and does not result in
any noticeable additional shimming. Note that if you have used the Varian 500 MHz NMR then
you do adjust Z1 and Z2. This demonstrates how parameters and methods cannot always be
readily transferred from one instrument to another.
• Close the lock/shim screen.
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SETTING UP THE EXPERIMENT AND COLLECTING DATA
• On the main screen, click on “Setup”. For most routine work you can choose “H, CDCl3” or
“C, CDCl3”. For other nuclei and solvents, choose “nucleus” and then “solvent” by clicking on
the appropriate boxes in the menu.
• The parameters of the spectrum can be changed by typing in the dialog box. For example
typing nt=8 <return> will change the number of scans from 1 (default value) to 8. The
parameter you may wish to change most frequently is nt. If you suspect your compound will
have resonances outside the usual range, increase sw (spectral width) from 6,000 to 10,000. In
each case, enter the parameter followed by an equal sign and the new value followed by
<return>. e.g sw = 13p
• Type su <return> to update the values
• type ga (go acquire) <return> to start the acquisition. You can follow the progress of your
experiment in the box located on the upper right hand side of the console, and if you have a
large number for nt, you can check the spectrum by typing wft (weighted fourier transform)
periodically, after a block of data has been collected (bs setting controls block size).
Type gettext to get a screen into which you can enter identifying information to be stored and
printed with your spectrum.
PROCESSING AND PRINTING THE DATA
• To save the spectrum, type: svf(‘filename’). You can retrieve the data later by typing
rt(‘filename’). Typing wft processes the FID and displays your spectrum. When the
spectrometer has finished scanning, your spectrum (or a portion of it) will be displayed.
• To process your spectrum type each command below separated by a space, then <return>
f
display full spectrum width (-1 to 12 ppm)
aph
autophase – automatic phasing of the spectrum
vsadj
adjust height of spectrum relative to the highest peak
dscale
display ppm scale on x axis
These commands display the full spectrum and automatically phase the spectrum.
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aph
• Assuming that you chose the proper parameters for your solvent, the instrument has set the
reference automatically. You can choose a particular window by typing wp=##p and sp=##p to
set the width and start of your spectrum (where ## is a number).
• There should be a cursor on the left hand side. If not, type ds (display spectrum). Dragging or
clicking the left mouse button adjusts the cursor position. Clicking the right mouse button
reveals a second cursor that can be dragged with the right hand mouse button to isolate a
chosen region. Clicking “expand” changes the window you have on the spectrum. Command f
will return to full expansion, or click the “full” button.
INTEGRATION OF THE SPECTRUM
The integral can be displayed in 2 modes, partial or full, by clicking on the “integral” button.
When in interactive mode, the second button of the lower menu toggles between No Integral,
Part Integral and Full Integral. Bear in mind that the button shows the next item in toggle
sequence, not the current mode.

Click on the integral button till an integral trace line appears.

Click resets button

To plot separate integral steps and compute their relative areas click on both sides of
the peaks to be integrated with the left mouse button. Move your pointer to the left of
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the first (most downfield) peak. Cut the trace there by clicking the left mouse button.
The integral trace to the left will become dotted (turned off) while that to the right will
remain solid (turned on). Move the mouse to the right of the peak and cut the trace
again. Now only that portion of the trace between the two "cuts" will be solid (on).
Repeat this procedure for all of the peaks, cutting the integral trace on either side of
each peak. This is called cutting the integrals. Only the peaks with a solid integral trace
will be integrated.
Helpful commands and hints
dc <return> corrects the baseline for drift and sometimes helps the integral.
The integral scale (vertical height) can be adjusted by dragging or clicking the middle mouse
button, or type in IS=##.
cz (clear zeroes) Cuts can be restored to the original full integral trace by
For peaks that are very close together, you may wish to integrate them as a single region.
Set Int button: place the cursor on top of one integral step, click “Set Int” button, and enter the
desired relative value for that integral. Command dpir will display the measured integral
regions.
Plotting THE SPECTRUM
To avoid plotting noise at the baseline, set the plot threshold.
• Clicking on the “Th” button displays a horizontal yellow line that can be moved with the right
hand mouse button to set a minimum threshold for “peak picking”. Only peaks above the
yellow line will be plotted.
Commonly used plot commands are:
pl
plot the spectrum
pscale
plot ppm scale
pir
plot integrals
pltext
plot text you entered
ppf
plot peak frequencies in ppm on the spectrum above each peak
pll
plot a line list, this is required for calculating coupling constants
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page
send data to printer and plot
For example, to plot your spectrum, including displayed integrals, scale, and a line list type
pl <space> pir <space> pscale <space> pll <return> page <return>.
For example, to plot your spectrum, including displayed integrals, scale, and text type
pl <space> pir <space> pscale <space> pltext <return> page <return>.
Note that text and a line list cannot be plotted at the same time as they occupy the same place
on the page and will overlap.
EJECTING THE SAMPLE AND LOGGING OUT
click on the “acqi” button then open “lock” panel
turn off spin and lock functions (important before ejecting or logging out!), and click on “eject”
remove your sample and click on “insert” to shut off the air
Close the acqi window
Type “exit” in the spectrum screen and let all the VNMR windows close. Then click the EXIT
button at bottom of screen. (Or, holding down the right hand mouse button at the bottom right
part of the screen drag down to “logout”. Hit return to confirm logout and you are done.) The
Login screen should appear for the next user.
THE STANDARD 1H NMR AND DECOUPLING EXPERIMENT.
Purpose: 1H NMR is a useful tool for the identification of compounds containing hydrogen,
including most organic compounds. The resulting spectrum contains useful information about
the structure of the molecule.
Typically, we have some idea about the composition or
structure of the molecule. In such cases, 1H NMR confirms our guess. At other times, we may
have other evidence to suggest a composition but little about the structure. In such cases, 1H
NMR is a versatile probe of the structure of an “unknown” compound. In this experiment, you
should obtain a spectrum of a standard compound (ethyl crotonate) and also one of your
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unknown. Your tasks are to 1) develop skill in obtaining well resolved, well phased 1H NMR
spectra, 2) become comfortable with the NMR spectrometer and 3) obtain information that will
help you identify your unknown.
O
5
1
ethyl crotonate
H
O
4
3
6
2 H
Procedure: A basic set of instructions for operating the 400 MHz Varian NMR spectrometer has
been provided above, except you will be choosing H,CDCl3 from the setup menu. The instructor
will review the instructions and demonstrate the basic 1H NMR experiment. After that, you are
on your own to develop your skills at NMR. It will take time to produce high quality spectra. A
known sample of ethyl crotonate is provided for you to practice with.
Once you are
comfortable that your spectrum has narrow line widths (resolution < 1.0 Hz), is well-phased,
and have printed and stored it, you are ready to move on to the proton decoupling experiment.
Make sure you have integrated each spectrum to help you in the structural assignment. Then,
return and repeat the same experiments on your unknown.
You may wish to experiment with several parameters that may affect your spectrum,
for example, changing the number of scans (enter nt = desired # of scans in multiples of 2) to
increase the signal-to-noise ratio. Rarely, there are resonances that may lie outside the
standard spectral width, in which case the spectral width can be increased with the sw
parameter. Increasing the delay time (seconds) between pulses by increasing d1, or shortening
the pulse width pw (microseconds) will lead to better integration in samples with long
relaxation times. NOTE: for sealed, degassed samples, relaxation times are often several
seconds, and it is wise to increase d1 and decrease pw to get meaningful integration results
(e.g., try d1=25 and pw=1; compare with standard settings).
Report:
• See separate comments on reporting NMR data! Assign all of the resonances in each
spectrum to specific protons in the molecules. You do not need to assign solvent impurities nor
the peak assigned to residual protons in the solvent unless you feel it is critical to your
discussion. It may be useful to print out expansions of spectral regions to display multiplets.
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• Organize your data in a table, and rationalize your assignments in a paragraph or two,
pointing out how the chemical shifts, splitting pattern, coupling constants, and integration of
each resonance supports your assignments. While you may find it useful to label the protons in
a drawing of the molecule on the spectrum, it is not sufficient to use this as your sole
justification for assignments. You may find it useful to consult a research article in a chemistry
journal for guidance in how to write your report.
• Submit clean, well-resolved and well-phased original spectra of the standard and your
unknown along with your report. This report should be combined with the report on the
homonuclear decoupling experiment.
HOMONUCLEAR DECOUPLING EXPERIMENT OF ETHYL CROTONATE
Purpose: In this experiment you will obtain the 1H spectrum of a standard sample, 5%
ethylcrotonate in CDCl3, under a variety of conditions to demonstrate the utility of
homonuclear decoupling in solving structural problems and simplifying spectra. Complex spin
systems lead to complicated multiplets in 1H NMR spectra. These spectra can often be
simplified by selectively irradiating one of the proton signals with a radiofrequency. The
resonance for this proton is removed (“saturation” due to equal populations in the two spin
states because of stimulation of rapid transitions between the two states) and any coupling to
that proton is missing from other signals. This allows us to determine which nuclei are coupled
to the nucleus that is irradiated, as well as simplifying complex multiplets. Application of this
technique to your unknown may help in clarifying either signal assignments or structural
interpretation.
O
5
1
H
O
4
3
6
ethyl crotonate
2 H
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PROCEDURE
Begin by recording an ordinary 1H NMR spectrum of 5% ethyl crotonate in CDCl3. Identify the
chemical shifts of the five sets of protons in the molecule based on chemical shifts and coupling
patterns.
To turn on the decoupler you will need to set the following parameters:
dm=‘y’ <return>
decoupler mode for first decoupler - yes
dmm=‘c’ <return>
continous
dpwr=25 <return>
decoupler power = 25).
Of these parameters, you may need to further adjust dpwr (suggested values 15 to 30). Choose
the resonance that you wish to decouple, expand around it, then place a single cursor (left
button) at the center of the multiplet you want to decouple, then type
sd <return>
set decoupler
su <return>
ga <return>
If the dpwr is not high enough, the irradiated signal will not be fully saturated (i.e. no signal)
and decoupling will not be complete.
After the acquisition, select full to see the entire spectrum.
Repeat this procedure to decouple other signals. At the minimum, you should select the
signal of the methyl attached to the C=C bond in ethyl crotonate. For your unknown, select at
least one signal to decouple. Some samples will give spectra that are more complicated than
others, so the necessity of doing repeated experiments may vary.
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Report:
This report should be combined with the report on the ordinary 1H spectrum and should
include:
• copies of any spectra, interpretation of couplings, and values for the coupling constants, J,
where they can be determined. To get values of the coupling constants, it is very useful to
print out line listings (pll page after setting threshold with the “th” button).
Use the decoupling experiment to determine 3JH3,H2
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THE STANDARD 13C NMR AND DEPT EXPERIMENT
Purpose:
13C
NMR is a powerful tool for the identification of compounds containing carbon,
including many organic compounds where the 1H NMR spectrum may be inconclusive. As in the
standard 1H NMR experiment, you should run the standard sample of ethyl crotonate as well as
your unknown. Your tasks include learning how to set the spectrum for measuring
13C
and to
assign signals.
PROCEDURE
Begin by shimming, then obtain a well-resolved 1H NMR spectrum of the sample. (If you have
previously recorded the 1H spectrum, you do not need to print and store it again.) The
shimming will carry over to give narrow lines in the
13C
spectrum. The same basic set of
instructions for operating the 400 MHz Varian NMR spectrometer provided above also work for
the
13C
experiment, except that you will be selecting C, CDCl3 during the setup for the
experiment. The biggest difference between the two experiments is that time needed for
signal acquisition for 13C is significantly longer. (Concentrated samples can help reduce the time
for the experiment but can introduce some line broadening.) As a result you may need to
acquire data for about 15 min to get decent a signal-to-noise ratio in your spectrum for a
sample containing about 10% compound in CDCl3. This is in addition to the time needed to
shim the magnet and to record a 1H NMR spectrum of each compound. It will not be necessary
to integrate the 13C NMR spectrum as integration is unreliable in 13C NMR.
As in the standard 1H experiment, you may wish to change some of the parameters. Most likely,
you will have to increase the number of scans. Try nt=64 for your unknown (concentration
about 10% by weight), although fewer may suffice. Use pw=5 (pulse width, microsec) and d1=5
(delay between pulses, sec).
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To start the experiment type:
nt=64
pw=5
d1=5
su
ga <return>
Report:
Combine this report with the results of the DEPT experiment, which can be run immediately
after acquiring the standard
13C
spectrum. Assign all of the resonances in the spectrum to
specific carbons in ethyl crotonate (indicate your reasoning and indicate if any uncertainty
remains). You do not need to assign solvent signals unless you feel it is critical to your
discussion. Use the DEPT experiment to aid in signal assignments. For your unknown, list the
observed peaks, along with the classification from the DEPT experiment, and any tentative
interpretation of carbon types based on the chemical shifts and DEPT data.
DEPT EXPERIMENT
Introduction: The 13C chemical shifts and intensities of aliphatic CH3, CH2 and CH carbons can
be similar. A distortionless enhancement by polarization transfer (DEPT) experiment is used to
determine how many protons are attached to each carbon in a
13C
spectrum. This can be of
considerable help in assigning signals or determining the structure of an unknown. In this DEPT
experiment, a total of four 13C spectra are recorded and processed to yield spectra where only
the CH3 carbons are shown, where only the CH2 carbons are shown and where any CH carbons
are shown. Carbon atoms that lack attached H’s do not appear in any of the DEPT spectra. The
actual experiment is a complex, multipulse sequence that fundamentally relies on one-bond
13C-1H
coupling for the information that is manipulated in the experiment. For instance, a CH
would give a doublet and a CH2 a triplet in an ordinary coupled spectrum, but the DEPT
spectrum just shows apparent singlets whose intensities vary. By comparison to a standard 13C
spectrum, one can often assign all of the resonances in a
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13C
spectrum. For the standard
sample, a concentrated solution of ethyl crotonate in CDCl3 (10%) is used to reduce acquisition
times for this experiment. The sensitivity of this experiment is actually higher than in a
standard 13C spectrum, so fewer scans can often be used.
Procedure: The DEPT experiment relies on a program already installed in the computer. Insert
the sample and lock it in the usual fashion. Select the C, CDCl3 parameters from the setup
menu. Record a standard
13C
spectrum, setting nt=128 so as to get a good signal to noise
spectrum.
Enter dept.
Set other parameters as follows:
pw90=14 <return>
90o pulse width for 13C
pw=14 <return>
pulse width used for 13C
pp=14 <return>
proton pulse width
pplvl=60 <return>
proton pulse power level
ss=2 <return>
steady state pulses before acquisition
j=140 <return>
average 1JH,C coupling constant.
Set nt=##/2 (DEPT is more sensitive than the ordinary 13C experiment), then ga.
For nt=64 the experiment will take approximately 15 minutes.
Four spectra will be acquired (first all C’s except quaternary; second just CHs third just CH 2,
fourth just CH3). After the first one is acquired, you can phase (aph) and adjust the vertical
scale (vs=##) and threshold (click on Th button). To get the full edited set, showing in one
display the spectra for all CH3, CH2, and CH, enter the following commands on one line
separated by spaces followed by the return key:
nll fp adept ds(1) vsadj vs=vs/4 vo=vs ho=0 full dss(‘all’) <return>
Enter pldept <return> to plot the set of edited spectra.
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The spectrum will appear as follows:
first line all C’s except quaternary
second line just CHs
third line just CH2
fourth line just CH3.
If your spectra have artifacts from poor subtraction, two likely errors are in saturation of the
lock signal (reduce lock power and gain) or a poor value for the decoupler pulse parameter pp.
Get help from the instructor if you suspect this is the problem.
Report: Combine this report with the report on the normal
13C
spectra. Your report should
include a clean copy of the edited DEPT spectra. In addition:
• Assign all of the resonances to specific carbons in the molecule. Warning: If your unknown
sample has a 13C signal with a large coupling to 1H, such as for a terminal alkyne where JCH ≈ 250
Hz, the standard setting of J = 140 will give an erroneous reading for that signal (e.g., may be
missing).
• Are there any resonances missing in the DEPT experiment?
responsible for the missing resonances?
• Are there any resonances that you can’t positively assign?
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Which carbon atoms are
EXPERIMENT 4
NOE DIFFERENCE EXPERIMENT OF 4-CHLOROANISOLE
Introduction: The nOe (sometimes “NOE” or “noe”) difference experiment provides a valuable
physical tool to make NMR signal assignments independently of couplings or chemical shifts.
Briefly, the nuclear Overhauser effect (nOe) is defined as the change in intensity of an NMR
signal when a second nucleus is irradiated with a radiofrequency to saturate the signal of the
second nucleus. The nOe effect can be quantified by a parameter h and is strongly related to
the distance (r6 distance dependence) between the nuclei. The nOe phenomenon derives from
the relaxation of nuclear spins due to the fluctuation in magnetic field felt by the nucleus from
a nearby nuclear magnet as the molecule tumbles in solution. When a continuous rf is applied
at the position of an NMR signal, the population in the two spin states for that nucleus become
equal, thus no signal will be seen for that nucleus (saturation). However, the population
difference for nearby nuclei become enhanced (a positive nOe). Since the nOe effect is
strongly distance dependent, it is a valuable tool not only for signal assignment, but also in
structure determination in showing the placement of groups nearby in space.
For this experiment, 4-chloroanisole will be used as the standard sample to test the
procedure before it is applied to your unknown. The 1H NMR spectrum of 4-chloroanisole is
very simple, consisting of a singlet for the CH3 substituent on oxygen and two approximate
doublets for the aromatic protons. (These peaks are not true doublets due to magnetic
nonequivalence.)
While the two doublets can be assigned as being ortho or meta to the
electron-donating methoxy group on the basis of chemical shift arguments, the nOe provides
independent verification. In the case of 4-chloroanisole, the CH3 group is closer to H2,6 nuclei
than to H3,5 so irradiation of the CH3 resonance will have a greater effect on the intensity of
the resonance for H2,6 than H3,5. This effect become readily apparent when two spectra are
recorded, a reference one without irradiation of the methyl and one where the decoupler is
employed to irradiate the methyl, followed by subtraction of the reference spectrum from the
methyl-irradiated spectrum. The intensity of the H2,6 resonance will be enhanced in the
methyl-irradiated spectrum and will result in a net positive signal in the difference spectrum.
The intensity of the resonance for H3,5 will actually be slightly weaker (negative nOe) in the
methyl-irradiated spectrum because of a relayed effect from H2,6 and will be upside down in
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the difference spectrum. If there were more distant nuclei, these would be unaffected and
show no net change in intensity.
H3C
O
H2
H3
Cl
4-chloroanisole
PROCEDURE
The spectrometer is equipped with a software routine (noedif) to run the nOe difference
experiment, but it generally does not give very good results. The procedure below works more
reliably. The general idea is to obtain two spectra under as similar conditions (instrument
settings, temperature, position of people in the room…) as possible, with the exception of a
different frequency setting for the decoupler. The decoupler is used to saturate a signal during
a delay before the sampling pulse, but is turned off during signal acquisition, so no decoupling
is seen in the spectrum, but the saturated signal will not appear and other signals may be
affected by an NOE.
Make sure first that you are operating in the “Experiment 1” section of computer memory by
using the command jexp1 <return> (join expt. #1)
Run (set pw=1, nt=2, ga, when done full, aph) and print a standard 1H spectrum.
Select which peak to irradiate and expand around that peak.
Place a single cursor (left mouse button) in the center of the peak and type sd (set decoupler)
Enter the following parameters. Hit the return key after each entry to make sure it was entered
correctly.
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dm=’ynn’
Decoupler mode for first decoupler
dmm=’c’
Decoupler modulation mode for first decoupler
dpwr=10
Power level for first decoupler with linear amplifier, 0- 63 dB
d1=3
First delay in seconds
ss=2
Steady-state transients
nt=16
Type gain? to find the current receiver gain (value in parentheses) and then enter a gain setting
of approximately half that value with gain=##.
Setup the experiment with su, then ga. (If you get an “ADC overflow” message, run it again with
a lower gain.)
When the acquisition is completed click full on the menu line to see the whole spectrum, and
phase the spectrum with aph.
Move the fid data from experiment 1 to experiment 2 by mf(1,2). (If experiment 2 is indicated
as inaccessible, type cexp(2) to create it, and try moving it again. If it indicates that experiment
2 is locked, type unlock(2)).
Change the decoupler offset to a position where no signal is affected, i.e., use the left cursor
and sd to reset the decoupler, preferably 1 ppm or so from the original position.
Run the spectrum again with su and ga
After the second spectrum is acquired, do not rephase it. Enter:
clradd
Clear the add/subtract buffer, Deletes the add/subtract experiment (exp5).
ai
Select absolute-intensity mode, to change to absolute intensity
spsub
to subtract this reference data in the add/subtract buffer (in experiment 5). (If
experiment 5 is indicated as locked or is otherwise inaccessible, type unlock(5), and try again.)
Then rejoin experiment 2, with jexp2, type wft, ai, and spadd (adds data to add/subtract
buffer).
To see the difference spectrum enter jexp5, then ds.
To see the full negative peak enter vp=100 and then adjust as needed. vp (vertical position)
adjusts the horizontal position of the baseline.
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The irradiated signal will appear as a large upside down signal, while other signals will be
mostly absent or show some “bumpiness” at their positions due to imperfect subtraction. Any
signal affected by an nOe will show a small real signal. You may have to play with the
expansion, vertical scale and integral scale settings for the portion of the spectrum of interest
(such as the aromatic region in 4-chloroanisole), but a signal with a real nOe will show a net
change in the integral line, while an unaffected signal will either be missing or just show some
bumpiness in the integral line with no net change. Integrate the NOE spectrum.
Report: As always, provide original copies of your spectra, both the standard 1H NMR spectrum
and the NOE difference spectrum. Calculate the % NOE effect. Briefly discuss how the nOe
difference experiment allows you to assign the two resonances attributed to the aromatic
protons.
At this point it is not necessary to perform a NOE experiment on your unkown. If you decide to
do so the signal(s) to select for irradiation will depend very much on what is needed to provide
further information for your structure, so you will need to have made some progress in
interpretation before running the nOe experiment, or you may have to return and try again
with other selections. If there are two signals close together, try lower power (dpwr) to be
more selective in the irradiation.
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EXPERIMENT 5 TWO DIMENSIONAL SPECTRA: 1H -13C CORRELATION (gHSQC) OF MENTHOL
AND UNKNOWN.
A broadly useful type of 2-D NMR experiment is one that provides a correlation
between hydrogen and carbon nuclei that are attached to each other. This is achieved via
multipulse sequences that take advantage of spin-spin coupling between 1H and 13C nuclei. In
the 2-D spectra, the signal intensity is represented in the vertical direction (contour lines) and
the x and y axes are proton and carbon frequencies. Cross signals in the 2-D spectra thus
indicate hydrogen and carbons that are connected to each other via coupling. For experiments
that select for large, one-bond coupling, the cross peaks indicate hydrogen and carbon nuclei
that are directly bonded. Such information is very useful because any signal that can be clearly
identified in one type of NMR spectrum (e.g., 13C) immediately allows assignment of a
corresponding signal or signals in the other type of NMR spectrum (1H). This is particularly
valuable when diastereotopic protons are present, because it allows ready identification of two
distinct signals for non-equivalent hydrogens attached to the same carbon.
There are several different experiments that can provide 1H -13C correlation. HETCOR,
or 1H,13C-COSY was developed first, but suffers from being a 13C -detected experiment that
requires long acquisition times (hours) due in part to the inherent insensitivity of 13C signals.
Inverse detection experiments detect the correlations via the more sensitive 1H channel and
can be done much more quickly. HMQC (Heteronuclear Multiple Quantum Correlation) is the
simplest form of inverse 1H -13C correlation techniques. The HSQC (Heteronuclear Single
Quantum Correlation) uses a more complicated pulse sequence but has become more popular
in recent years. Both HMQC and HSQC are based on the occurence of one-bond 1H -13C
couplings of the magnitude of about 120-180 Hz. Another useful experiment is HMBC
(Heteronuclear Multiple-Bond Correlation), which suppresses signals from the one-bond
coupling and is selective for two- and three-bond 1H -13C couplings of about 4-14 Hz. The most
recent developments are pulsed-field gradient (PFG) experients that accelerate data gathering
even more. (The field gradient destroys the coherence of the precessional motion of the
nuclei, thus allowing more rapid rf pulsing than allowed by ordinary relaxation processes.)
The first technique that will be described here is a PFG HSQC experiment with decoupling.
The 1H -13C cross peaks can be made effectively into singlets, showing no coupling to 13C in the
23
1H
dimension, if 13C decoupling is applied during signal acquisition, so such HSQC spectra are
typically very easy to interpret. Carbons that have protons attached will show cross peaks that
correlate with the 1H frequencies of the attached protons. Carbons that do not have protons
attached will not show up. (A disadvantage of decoupled spectra is that if decoupling is not
complete for some reason, it is possible that a peak will be too broad and weak and might be
missed—a coupled gHSQC spectrum can be obtained by setting dm=’nnn’ along with setting
other parameters as described below.)
The success of the experiment does depend on a delay parameter that is related to the
C-H coupling constant, which has been preselected as 140 Hz; 140 Hz is usually suitable for sp 3
and sp2 C-H bonds, but will fail for sp C-Hs in terminal alkynes where J≈250 Hz. Actually, several
parameters have been pre-set because optimizing the experiment can be a lengthy process,
while obtaining a spectrum once the parameters have been established can be quite fast
(around 15 minutes for a concentrated sample). Preset parameters include the settings for the
gradient pulse; other parameters have default values that may work, but the recommended
current settings are listed below.
Steps in the gHSQC Experiment:
As in the usual experiments, insert the menthol sample, lock and shim.
Obtain a routine 1H-NMR and 13C-NMR to insure your sample is giving a good spectrum.
Type jexp1 (join exp 1) <return>, then acquire one scan (setup–H,CDCl3, nt=1 ga) of an
ordinary 1H 1-D experiment. Set the cursors about 0.5 ppm beyond the last proton resonances
at both ends of the spectrum, then movesw (move spectral width).
Re-acquire the 1H spectrum (nt=1 ga) and phase it (aph). Move the parameters to another
experiment (such as #3) with mp(1,3), then join that experiment with jexp3. This will set the
spectral width in the 1H dimension in the gHSQC spectrum.
Set up the gradient HSQC experiment with gHSQC return. (If a message such as “p.s.g.
aborted” appears and the pulse sequence is not shown in the display window, type gHSQC
24
again.) Turn off the spin in the ACQI window, and make sure the sample remains locked on the
deuterium solvent signal. If it unlocks, increase lock power or gain until it is locked.
To make sure the gradient field is available to the experiment, type pfgon=’nny’.
Set the following parameters, which should work for the unknown samples that are fairly
concentrated (≈ 10%):
nt=2
or a multiple of 2
ni=128
number of iterations (points in the t1 dimension); use
ni=256 if greater resolution in the 13C frequency is
desired
d1= 1.0 second
tpwr=60 dB
proton transmitter power
pw=14.0 microseconds
current value for proton 90-deg pulse width with
tpwr=60
pw90=14.0 microseconds
current value for proton 90-deg pulse width
pwx=14.0 microseconds
current value for carbon 90-deg pulse width with
pwxlvl=60
pwxlvl=60 dB
carbon transmitter power
dmf=8200
decoupler modulation frequency
dpwr=45 dB
decoupler power
The default parameters for spectral width (sw1) and decoupler offset (dof) in the 13C dimension
are set to cover from about -10 to 160 ppm; these parameters are in Hz. If you want to change
them, for instance if you have a proton-bearing carbon such as an aldehyde that appears
beyond that range, you should perhaps return to another experiment, recall the carbon
spectrum, and use the spectral width and transmitter offset (tof) from that experiment to set
the new values of sw1 and dof. If you use a wider sw1, you should probably also use ni=256.
The default value for the 1H -13C coupling constant is j1xh=140. This works for most cases, but
wouldn’t work for an alkyne C-H (J of about 250 Hz), and may not be high enough for an
25
occasional aromatic or vinyl C-H (benzene is 160 and ethene is 156 Hz, but pyridine C2-H is 178
Hz). In these cases type j1xh=250 su enter
Start acquisition by typing ga (ga will display the 1-D results of each pulse).
Data Processing:
There are a lot of adjustments that can be applied to 2-D data—too many to describe here. If
you used the standard conditions, the following description should work.
When the experiment has finished acquiring data, type wft2da (weighted Fourier transform of
2-D data, automatic). It would be wise to gettext, label the experiment, and save
(svf(‘filename’)) before proceeding much further. You can save it again if you like your final
processed spectrum better.
A contour plot should be displayed. If not, type dcon or dconi (display contour interactive),
where dconi lets you interactively adjust the display. (If you get the message “scale outside
boundaries, adjust sc wc”, then type full dconi) You can adjust the vertical scale with vs+20%
and vs-20% menu buttons (recommended!), or with the middle mouse button, or by manually
changing the parameter vs2d by typing vs2d=xxx, where the smaller the “xxx”, the lower the
vertical scale. You should try different vertical scale adjustments until you find a setting that
shows all of the real cross peaks without too much noise (horizontal or vertical streaks or just
background noise). (If you change some settings, you may need to retype dcon or dconi.)
When the display is as desired, set th=0 (threshold of zero), and plot the results by typing plot
<return> which gives you the contour plot with projections of the peaks along both the 13C and
1H
dimensions. Also try plotting the spectrum using rplhxcor. From decoupled HSQC spectra,
these projections will ideally be singlets in both dimensions, but broad due to the reduced
resolution from ordinary 1-D spectra (fewer data points). In the default mode of running HSQC
spectra, methyl and methine signals give positive projections and methylenes give negative
projections (i.e., the signals have the opposite phase). You can also plot just the contour
26
spectrum with pcon, or plot just negative or positive peaks with pcon(‘neg’) or pcon(‘pos’)—
that may remove some extraneous noise.
You can expand the part you want to display by drawing a box around the area with two
cursors then expanding that region. To do so, after dconi, click in the lower left with the left
mouse button and upper right with the right mouse button, then click the expand button. You
can also find the positions of the cross peaks using the cursors.
Repeat the experiment on your unknown
Reporting data:
HSQC data can be reported by listing 13C peak positions from an ordinary 13C spectrum and
then listing after them the correlated 1H signals as determined from the HSQC spectrum.
TWO DIMENSIONAL SPECTRA: 1H -1H CORRELATION (COSY)
As in the usual experiments, insert sample, lock and shim. Type jexp1, then acquire one scan
(nt=1, ga) of an ordinary 1H 1-D experiment. Box the cursors about 0.5 ppm beyond the last
proton resonances at both ends of the spectrum, then movesw (move spectral width).
Re-acquire the 1H spectrum (nt=1 ga) and phase it. Move the parameters to experiment 3 with
mp(1,3), then join that experiment with jexp3.
Use the default parameters that come with the gCOSY command after moving the proton
spectrum parameters, but set the current 90-deg pulse to its current value, i.e., pw=14 and
pw90=14. nt=1 or 2 should suffice to get good signal-to-noise.
To run: type gCOSY nt=1 ga <return>
27
The experiment should take < 5 minutes. If the time in the status window is > 10 minutes, there
is a problem.
Processing the spectrum:
When the experiment has finished acquiring data, type wft2da (weighted Fourier transform of
2-D data, automatic). It would be wise to gettext, label the experiment, and save
(svf(‘filename’)) before proceeding much further. You can save it again if you like your final
processed spectrum better.
A contour plot should be displayed. If not, type dconi (display contour interactive).
You will need to use the ± 20% buttons to adjust the height of the contours. Readouts for
parameters such as the following appear in the lower part of the screen:
• cr shows the current cursor position.
• cr1 shows the current cursor position along the first indirectly detected dimension.
• delta shows the cursor difference.
• delta1 shows the cursor difference along the first indirectly detected dimension.
• vs2d shows the vertical scale of the display.
• vsproj shows the vertical scale of the trace or projection.
To plot the spectrum type plot <return>
References
The experiments described herein are based loosely on those in S. Berger and S. Braun 200 and
More NMR Experiments Wiley-VCH 1998, the Varian Manuals that accompany the NMR
instrument (NOE Difference, COSY, DEPT) or from B. L. Jensen and R. C. Fort, Jr. J. Chem. Ed.
2001, 78, 538 (VT)
28
Routine Varian NMR Commands
At the beginning I suggest using the MENU button commands as much as possible. Less to
remember and you avoid the possibility of accidently typing a command that takes you into
UNIX land.
Obtaining a spectrum
ga
su
aa
nt
starts data acquisition and performs weighted fourier transformation
Submit a setup experiment to acquisition (M), Sets up the system hardware
to match the current parameters but does not initiate data acquisition.
Everything that su does is also done by go, ga, and au
abort acquisition
Number of transients, up to 109. Another way of thinking about it is the
number of repetitions or “scans” performed
Printing
The Varian software plots, not prints, a spectrum
pl
pscale
pir
page
pll
pltext
pap
ppf
pps
put the spectrum on the plot
plot the scale in ppm at the bottom of the spectrum
add the integrated area below each integral region
sends the plot to the printer
prints a line list of Hz and ppm for each peak above the threshold
adds what was typed (annotation) into the gettext window
adds annotations and the parameters for the experiment to the plot
plots the chemical shifts in ppm above each peak
plots the pulse sequence used for the experiment
typical string commands, leave a space between each command
pl pscale pir page
plots the spectrum, the ppm scale, and integrated areas
pll page
plots the line list on a separate piece of paper
pl pll pscale pir page plots all the above on one piece of paper
To change the appearance of the plotted spectrum
sc=x
wc=x
vp=x
start of the plot is x mm from the right edge of the paper
width of the plot is x mm
letter size paper is about 216 mm by 279 mm
sets the baseline of the spectrum x mm from the bottom. Useful command for
NOE where negative lines are obtained. Limits are -200 to +200 mm
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Mouse Functions
Left
Middle
Right
select buttons, decrease values (e.g. lock gain), cuts integrals in resets
adjust height of spectrum, integrals
increase values
Working with the spectrum
bc
dc
cz
vsadj
th
rl
f
io
ds
aph
adjusts the baseline if it is drifting
spectral drift correction – use if the integral line is drifting down or up
clear integral reset points
adjust the vertical scale of the peaks relative to the largest peak
threshold use to select which peaks will be detected for the line list,
does not affect integration
reference line use to set the TMS peak at 0 ppm
shows the full spectrum, can use if you have expanded a region and want
to go back to the full spectrum
integral offset – move the integral lines with the middle button
display spectrum – try if the spectrum disappears
automatic phasing of the spectrum, if the shimming was optimized, it should
be all you need to do to phase the spectrum
30
MAIN MENU BUTTONS
31
SETUP button takes you into the different experiments
Selecting Setup in the main menu activates the Setup menu, which is used to set up new NMR
experiments. Additional menus can be displayed from the Setup menu to select the nucleau,
solvent, and pulse sequence.
Button
Description
H1, CDCL3
C13, CDCL3
Nucleus, Solvent
Set up a standard proton experiment with CDCl3 as solvent
Set up a standard carbon experiment with CDCl3 as solvent
Display the Nucleus Selection menu to set up a standard experiment
using menus to select nucleus and solvent.
Display the 1D Pulse Sequence Setup menu to select a sequence from
a list of 1D and 2D pulse sequences
Sequence
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Interactive 2D Color Map Display Main Menu
The buttons the Interactive 2D Color Map Display Main Menu function as follows:
Box - The first button is labeled Box or Cursor, depending on the display mode you are in. If
labeled Box, you are in the cursor mode, and this button changes the display to he box mode
with two pairs of cursors. Cursor - If labeled Cursor, you are in the box mode, and this button
changes the display to the cursor mode with one pair of cursors.
Trace Selects the trace display mode.
Proj Displays the Interactive 2D Display Projection Menu, see below.
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Expand The fourth button is labeled Expand or Full depending on the mode you are in. If
labeled Expand, you are in the box mode and this button expands the area between the
cursors.
Full- If labeled Full, you are in the cursor mode and this button displays the full area.
Redraw Repeats the last 2D or image display with current parameters.
Plot Plots the current trace.
Peak Displays the Interactive 2D Peak Picking Main Menu—ll2d program.
Return Returns to the previous menu.
Interactive 2D Display Projection Menu
The buttons the 2D Display Projection Menu function as follows:
Hproj (max) Displays a horizontal projection of the maximum intensity at each frequency.
Hproj (sum) Displays a horizontal projection of the summed intensity at each frequency.
Vproj (max) Displays a vertical projection of the maximum intensity at each frequency.
Vproj (sum) Displays a vertical projection of the summed intensity at each frequency.
Plot Plots the current projection.
Cancel Returns to the Interactive 2D Color Map Display Main Menu, see above.
Controlling the Display with the Mouse
The left and right mouse buttons are used to move cursors, the center button to adjust the
vertical scale of traces, projections and contour maps, as well as to adjust the threshold in the
color bar. The cursors can be used to select regions for expansions of the display. The cursors
can also be used to select positions to “mark” using either the mark command or the
ll2d('mark') command. Both commands display and record spectral frequencies, maxima,
intensities, and volumes. ll2d('mark') is recommended, however, because it also allows for
interactive display and editing of mark locations.
Left Mouse Button
The left mouse button adjusts the position of the 2D cursor. The corresponding frequencies are
displayed at the bottom of the graphics window. Both the horizontal and vertical cursors move
if the left mouse button is pressed within the 2D display box.
Above and below the box, only the vertical cursor can be moved; at the left and the right of the
box, only the horizontal cursor. In addition, holding the mouse button down and then moving
the mouse moves the cursor with the mouse.
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Center Mouse Button
The function of the center mouse button depends on the location of the cursor:
•If the cursor is within the 2D display box, for color map and contour displays, if there is no
intensity at that point, the center button changes vertical scale to show intensity at that point.
If there is intensity at the point, the center button changes the scale to show no intensity, then
changes the parameter vs and redraws.
•If the cursor is near an active trace and active horizontal or vertical projection, pressing
the center button changes the vertical scale of trace or projection, so that spectrum goes
through the current mouse position.
•If the cursor is near the color/grayscale bar and in the color mode, pressing the center
button sets the threshold to remove low intensity peaks. If in the grayscale mode,
pressing the center button sets the grayscale intensity (the right button adjusts contrast).
Right Mouse Button
A second cursor pair is displayed with the right mouse button. The second pair can be
moved in exactly the same way as the first pair, and is used to select a box within the 2D
display. The right mouse button also switches the display into the box mode, the same as
clicking on the Box button in the menu.
Changing the Display
The user interactively modifies the display through selecting buttons on the menus,
pressing the buttons on the mouse, and moving the mouse. If desired, commands and
macros can also be typed in at any time.
Displaying Traces
The Trace button can be used to display a trace at the current position of the first horizontal
cursor. The left mouse button still moves the cursor, and different traces will be selected and
displayed accordingly.
The vertical scale of the trace can be adjusted with the center mouse button, by placing the
mouse arrow above the spectrum at the requested height and then pressing this button. Do
not press the center button at this time within the 2D display box.
The trace mode can be left by displaying a box with the right mouse button, or by selecting any
other display mode.
Displaying Projections
With the Proj button, you can display projections from the 2D Display Projection Menu.
Projections can be made in horizontal and vertical direction, and are available as the sum or
35
maximum of the data. Select one of the four available modes or cancel the operation with the
Cancel button. Again, the center mouse button adjusts vertical scales.
Expanding the Display
Once a box is selected, you can click on the Expand button to obtain an expanded display.
Alternatively, if you are in the one cursor mode, you can click on the Full button to display the
full 2D display. In this case, the two cursor pairs mark the last expanded region and the
Full/Expand button toggles between the full and expanded mode.
An alternative way to select an expansion is to type in new values for the parameters sp,
wp, sp1, and wp1 (e.g., sp=100 wp=50 sp1=100 wp1=50), then use the Redraw button to
redisplay.
Setting the Vertical Scale
The vertical scale can be adjusted in a number of ways. If a peak is expected at a certain
position in the spectrum but is not visible, the mouse arrow can be moved to that position and
then the center mouse button pressed once. This selects a new vertical scale, so that the
intensity at that point is by a factor of 2 above the threshold, and the display is redrawn. Be
careful in this mode not to queue up several redraw operations.
Adjusting the Threshold
If noise is visible at a certain position in a spectrum, but should be suppressed below the
threshold, move the mouse arrow to that position and press the center button. A vertical
scale is calculated so that this intensity falls by a factor of 2 below the threshold, and again the
spectrum is redrawn. If the peak is visible but is not a factor of 2 above the threshold, clicking
on the center button increases vs2d.
36