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Two dimensional experiments with VNMRJ

E.Alvarado 9/15/06

General remarks
This manuscript is designed to provide only the minimum information required to perform the
most common routine two dimensional experiments with VnmrJ in the NMR Facility at the
University of Michigan. It doesn't include detailed information about optimization of parameters
or about the NMR techniques. The user must be aware that further optimization, before and
after acquisition is possible. However, Vnmrj is very complex, with many more options and
commands than a casual user would want to know, and making this manuscript more
comprehensive would render it tedious for such kind of user. Extensive information can be
found in the references.

Acquire and optimize a proton spectrum before proceeding. If you are doing a 1H - 13C
correlation it is also advantageous (but not required) to have a carbon spectrum. Setup your
proton spectrum in workspace 1 (experiment 1), your carbon in workspace 2, and your 2Ds in
workspaces 3, 4, etc. That way, you can easily switch from one spectrum to another for
examination, referencing, etc. Please note that in general, in our Facility the Inova 500 is the
preferred instrument for almost all 2D experiments. This is because the default probe in the
Inova 500 is an “inverse” probe, i.e. it is optimized for proton detection and has better
sensitivity and lineshape for protons. Nevertheless, it is still possible to get 2D correlations on
the 400 and 300 MHz instruments, but they will take longer or require more concentrated
samples. As a rule of thumb, at the same concentration you will require at least double the
number of scans on the Inova 400 and probably four times on the Mercury 300 compared to
the Inova 500. Note however that the probe on the Inova 500, tuned by default to 1H and 13C in
CDCl3, is somewhat sensitive to solvent changes. When doing a 2D on a solvent like D2O,
DMSO or methanol, it may be necessary to retune the probe to get the best results. Please
ask the facility personnel about retuning the probe; do not attempt to do it yourself without
authorization.

Acquisition
Start by running a quick 1D 1H spectrum in workspace 1. Narrow the spectral width to the
region of interest leaving about 1 ppm margin on both sides (select the region using the
cursors and type movesw) and take a new spectrum with the new parameters. Reference it
with TMS or the standard that you use. You can save it to disk now. Copy it to workspace 3
(mf(1,3)). If desired, you can go to workspace 2 and get a 13C spectrum. Save it too.

Go to workspace 3. From the Experiments menu select the experiment that you want. In
general, gradient experiments are preferred as they are faster to acquire and produce cleaner
spectra, but they are also slightly less sensitive than the non-gradient versions. If your sample
is concentrated enough to get a good proton spectrum with only 1 or 2 scans (at least 10 mg
for a medium molecular weight organic compound) then choose a gradient experiment. If your
sample is so dilute that you need 8 scans or more, then selecting a non gradient experiment
may give you better sensitivity, but keep in mind that non gradient experiments require at least
8 scans to suppress unwanted signals. With less than 8 scans you will see artifacts in non
gradient experiments. Gradient experiments start with the letter “G”, i.e. Gcosy, Ghsqc,
Ghmqc, Ghmbc. The Mercury 300 does not have a gradients unit: select a non gradient
experiment. See the experiment selection table at the end of this manuscript for help in
selecting an experiment.
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In the Acquire, Defaults panel select an appropriate number of Scans per increment and a
Number of increments. The minimum numbers should be 2 and 128 respectively. The number
of scans should be determined by the concentration of the sample. With at least 10 mg of a
medium molecular weight sample, a Ghsqc should give a decent spectrum in 15 minutes in the
Inova 500 (double that time in the Inova 400). A gCOSY takes only about 5 minutes in the
Inova 500. The Show Time button displays the total time the acquisition will take. If you want
to increase the number of scans (to get better signal to noise), it may be advantageous to
increase the number of increments instead. This will also improve signal to noise but
additionally it will improve digital resolution in F1. So for example, if you have nt=2 ni=128,
set ni=256 instead of nt=4. The acquisition time will increase by the same amount in both
cases; it will double.

In the Acquire, Defaults panel you can also change the spectral width if not already done or, in
the case of a heteronuclear experiment, the C13 Spectral Width, but the default value should
be fine for most typical organic compounds. Set “Linear prediction in t1” to “default”. See
Figure 1 for examples.

Figure 1. Acquisition panels for Gcosy and Ghsqc experiments.

Other parameters that may be changed are found in the Acquire, Pulse Sequence panel. The
parameters shown depend on the pulse sequence used (Figure 1). For example, you can turn
on solvent presaturation, useful with non-deuterated solvents to reduce the huge solvent peak
(notice that a critical parameter is missing in the panel, satfrq, the frequency of the solvent
peak). The Steady State option enables a section in the pulse sequence that destroys residual
transverse magnetization before each pulse. The purpose is to reduce t1-noise (the vertical
ridges of noise in strong peaks). In practice, this technique sometimes helps but sometimes it
makes the spectrum worse. In general, don't enable it unless you already took a spectrum and
it has strong t1-noise. In this case, it will also help if you increase the relaxation delay d1 and
the number of increments.

In the case of heteronuclear experiments you will find other parameters in this panel that can
be optimized if you want. Look in Varian's “VNMRJ Liquids NMR” reference manual and in the
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tables at the end of this manuscript for more information.

Figure 2. Pulse sequence parameters for Gcosy

In general, the default parameters are good enough for most routine analyses. You are
advised not to change them unless you have already done several 2D experiments and have a
good idea of what you are doing or unless you already took a 2D spectrum of this sample and
want to improve the results.

Figure 3. Pulse sequence parameters for Ghsqc

Start the acquisition with go or by clicking the Acquire button. Don't forget to save your
spectrum after it is finished. After the acquisition starts, you can go to a new workspace, setup
a new experiment and start it. The new experiment will be placed in a queue and started right
after the previous one is finished.

Processing
When processing on a workstation, be aware of a “feature” of vnmr. Normally, when a user
double clicks on a filename in Vnmrj, the FID is loaded and “autoprocessed”. During
autoprocessing the FID is not only Fourier transformed but also automatically referenced
according to the solvent. If the spectrum was properly referenced before it was saved, the new
reference set by the program will probably be wrong! If you want the original reference to be
kept, then autoprocessing must be disabled. You can do this with doprocess[1]='n' or by
deselecting “Process data on drag-and-drop from locator” from the Utilities, System Settings,
Display/Plot menu. Then when you double click on a filename or open a file from the File
menu the FID will be loaded but not altered or transformed, so you will have to do the Fourier
transformation, phasing, etc, after loading it.
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Before continuing, please take a few minutes to familiarize yourself with the display icons:

Most of the processing operations are controlled from the Process, Process panel (See Figure
4 for reference). Two-dimensional spectra are usually preprocessed to enhance their
appearance. Linear prediction is applied along F1 to reduce FID truncation artifacts and a
weighting function is applied to both dimensions to improve lineshape. Click on [Set default t1
Linear prediction] to calculate its parameters. Click on [Full 2D Transform] to do the Fourier
transformation. The 2D spectrum should be displayed. If you get an error message similar to
“scale outside boundaries...” go to the Process, Display panel and click on “Screen position”
Full. Homonuclear spectra can be symmetrized along the diagonal with the foldt macro to
help eliminate t1 noise but this can also reduce real peaks, so beware. If the baseline of the
traces is not flat, clicking on [BC F2] or [BC F1] buttons in Process, Display panel may help to
get a better looking spectrum by applying a baseline correction along the desired dimension.

Figure 4. Process panel for Ghsqc

Referencing. If you have the proton spectrum in workspace 1 and it is properly referenced,
you can set the proton dimension of your 2D to use the same reference with mref(1,3). The
second dimension can then be referenced with reff1. Alternatively, you can choose a cross
peak and use it to reference both dimensions. Expand the region around a cross peak whose
chemical shifts in both dimensions are known and place the cursor in the center (Figure 5). It
is better if you have real contour displayed (Process, Display, “2D Contour Display” buttons).
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In the example below the chemical shifts should be 5.030 ppm in F2 (proton) and 68.01 ppm in
F1 (carbon). To set these chemical shifts type: rl(5.03p) rl1(68.01d). Or, enter the
appropriate values in the left side column of the Process, Display panel.

Figure 5. Setting the reference in a 2D spectrum.

Phasing. Some 2D spectra like COSY, CIGAR and HMBC are transformed in “magnitude
mode”. The peaks in these spectra are always positive and do not require further adjustment.
Other spectra, like HMQC, HSQC and NOESY, are acquired in “phase sensitive” mode and
may contain both positive and negative peaks. If your spectrum contains cross peaks that look
like comets, i.e. the cross peaks have tails on one or two of their sides, it probably requires
phasing (See Figure 6).

NOESY spectra generally require phase correction. To phase a NOESY spectrum, first make
sure F2 (the vertical trace in Varian's default display mode) is accurately phased. Click on the
[FT 1D-1st Increment] button on the Process, Transform panel and phase this 1D spectrum
carefully; increase the intensity a lot and make sure the baseline around the peaks is phased
(if the baseline is not flat, correct it with “bc”). Retransform the 2D spectrum. Additionally,
NOESY spectra usually require a phase correction in F1 (the horizontal trace). Apply the
correction with rp1=rp1+45 and then redisplay the spectrum with the “redraw screen” icon.

To phase a other 2D spectra like HMQC or HSQC or to fine adjust a NOESY you must select
two horizontal slices, one containing a peak on the right side of the spectrum and the other
containing a peak on the left side. Notice that, due to a bug, with version 1.1D of Vnmrj the
procedure that follows works only when the complete 2D spectrum is displayed; i.e. it does not
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work with expansions. Vnmrj 2.1B does not have this problem. If you get the error “Cannot
phase this data”, type pmode='full', retransform the spectrum and start the phase
correction procedure from the beginning.

In the procedure that follows, it is imperative that you understand the results expected for a
given experiment and phase your spectrum accordingly. For example, individual cross peaks
in both HSQC or HMQC experiments should be completely positive or completely negative but
some cross peaks can be positive while others may be negative in the same spectrum (Fig 6);
cross peaks in Dqcosy experiments will have both positive and negative components (for
example, a peak in a doublet will be positive while the other may be negative); and in a
NOESY experiment the diagonal should be negative, positive cross peaks indicate positive
NOE's and negative cross peaks are either negative NOE's or exchange correlations. For
NOESY spectra, the opposite convention is also used, ie. the diagonal is phased positive. In
this case, what is important is the relative sign of the cross peaks, their absolute sign is
irrelevant.

Figure 6. A gHSQC spectrum that is out of phase (left) and the properly phased spectrum
(right). Warm colors represent positive cross peaks and cold colors represent negative ones.

You should also know that the weighing functions normally applied to these experiments can
result in artifacts that appear as negative or positive ridges on both sides of diagonal and cross
peaks. While these artifacts are unavoidable, they may be minimized by proper adjustment of
the weighing functions.

1. Click on the phase correction icon

2. Move the cursor until you get to a horizontal trace with a strong peak upfield
3. Click on the “select upper right peak” icon
4. Move the cursor until you get to a horizontal trace with a strong peak downfield
5. Click on the “select lower left peak” icon
6. Click on the phase correction icon again. If you get the error “Cannot phase this data”,
type pmode='full', retransform the spectrum and start the phase correction
procedure from the beginning.
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7. Click on the “select upper right peak” icon and click and drag on the rightmost portion of
the spectrum to phase it. If there are no peaks downfield do not click or attempt to
phase the left side of the spectrum.
8. Click on the “select lower left peak” icon and click (but do not drag) the rightmost
portion of the spectrum (this prevents Vnmrj from changing the phase correction found
in step 7). Then click and drag on the leftmost side of the spectrum to phase this side.
You can repeat steps 7 and 8 to verify (or fine adjust) that whole spectrum is in phase.
9. Click on the return icon twice to get to normal 2D display mode
10. Check that your spectrum is properly phased along the horizontal and repeat the
procedure to make adjustments if necessary.
11. Check the phase correction along the vertical axis. If it shows signs of being out
of phase, click on the “rotate along diagonal” icon and repeat this procedure.
When you are done, click on the rotate along diagonal icon again to go back to
the usual (Varian's) display convention.

Plotting
After you have selected and zoomed in the region of interest, you must choose the intensity
threshold. Use the “increase intensity” or “decrease intensity” icons to set an intensity just
above the noise level. You can also use the color scale on the right of the spectrum for this
purpose (click with the middle button). After this you must select how many contours you want
displayed and their separation. Use the command dpcon to do it. Dpcon takes two
arguments; the first is the number of contours and the second is the separation (a number,
usually between 1.0 and 2.5). If you are plotting a large area of the spectrum the cross peaks
will be very small and you won't be able to see too many contours (try 5); if you want to expand
a small region you will get a better looking spectrum if you use many contours (try 12 to 16).
Start with dpcon(5, 1.5) and change the parameters until you get a spectrum in which all
your peaks are well defined, with contours that go from the base (just above the noise level) all
the way to the top. You will use these parameters in the plot command.

In order to differentiate positive from negative cross peaks, spectra are plotted in black and
gray. The default is black for positive peaks and a shade of gray for the negative ones; the
exact “color” or shade is selected with the color command. If you get everything in black
type “maxpen=8” and try again.

For homonuclear experiments the plot command is, for example, plcosy(5, 1.5, 1) where
the first two parameters are the ones you found with dpcon and the third is the workspace
number where the high resolution 1D proton spectrum is located.

For heteronuclear experiments the plot command is, for example, plhxcor(5, 1.5, 1, 2)
where the first three parameters are the same as in the plcosy example and the fourth is the
workspace where the high resolution 1D 13C spectrum is located. If you don't have a carbon
spectrum omit the fourth parameter. It is also possible to “extract” a low resolution carbon
spectrum from the 2D (a projection) and use it to plot it alongside the 2D.

Notice that the plhxcor macro supplied by Varian requires that all spectra had been acquired
on the same spectrometer, otherwise the chemical shifts won't match. This makes sense as a
1D spectrum measured at a different field may look different. It will certainly be the case for
proton spectra because their splitting patterns are so sensitive to field strength. In our lab this
is inconvenient as the most sensitive spectrometer configuration for C13 is the Inova 400 while
the most sensitive (and recommended) for heteronuclear 2D correlations is the Inova 500. To
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provide more flexibility, the macro plhxcor was modified in all the computers in the lab so that
it is now possible to plot a C13 spectrum collected on the Inova 400 along a 2D collected on
the Inova 500. Keep in mind that this is not formally correct as you are displaying and
comparing spectra at two different fields. However, in the case of natural abundance C13
spectra and other nuclei lacking homonuclear coupling, and for appearance purposes, it may
be acceptable.

Experiment selection guide

Homonuclear correlations

Name Remarks Adjustable parameters

Cosy, Gcosy Classical correlation experiments
Magnitud mode. Very easy to run.
Variations are possible: COSY-45, COSY-LR.
Dqcosy, Double Quantum filtered Correlation Spectroscopy.
Gdqcosy Phase sensitive. Suppresses singlets in diagonal. Less
sensitive than COSY but higher resolution.
Measurement and analysis of coupling constants is
possible.
Noesy Dipolar (through space) correlation experiments. Mixing time can be 0.1 s to 0.3 s
Phase sensitive. Diagonal peaks are negative, positive for large molecules or 0.3 s to 0.8
cross peaks are positive NOEs, negative cross peaks s for small molecules.
are negative NOEs or chemical exchange correlations.
Cosy-type artifacts may appear between coupled
protons as antiphase cross peaks.
Roesy Rotating Frame Overhauser spectroscopy. Mixing time can be 0.1 s to 0.3 s
Phase sensitive. Diagonal peaks are negative, all direct for large molecules or 0.3 s to 0.8
NOEs are positive cross peaks, indirect NOEs are s for small molecules.
negative and chemical exchange correlations appear as
negative cross peaks. Tocsy-type artifacts may appear
as antiphase cross peaks. Use Roesy instead of Noesy
when the molecular weight is above 1000.
Tocsy Total Correlation Spectroscopy (HOHAHA; homo- Mixing time 30 - 500 ms.
nuclear Hartmann Hahn).
Phase sensitive, correlates a proton to all other protons
in a spin system. Very useful to see individual residues
in peptides, oligosaccharides and some other organic
compounds.

Heteronuclear correlations

Name Remarks Adjustable parameters

Hsqc, Ghsqc, Heteronuclear single bond correlation. The average value of the one
Hmqc, Ghmqc Phase sensitive. Cross peaks in Hsqc are multiplicity bond coupling constant can be
edited by default; CH and CH3 are positive and CH2 optimized if necessary. The
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Name Remarks Adjustable parameters

are negative, so you get the equivalent of a DEPT default is 140 Hz.
experiment at the same time. Can also be used to
obtain 15N chemical shifts at natural abundance. Hsqc
gives higher resolution and is preferred over Hmqc.
Hmbc, Ghmbc Heteronuclear Multiple Bond Correlation. The average value of the 2,3 bond
Phase sensitive. Shows correlations through 2 and 3 coupling constant can be
bonds (C–C–H) and (C–C–C–H). Useful for optimized. The default is 8 Hz.
assignment of quaternary carbons or tertiary amines.
Cigar2j3j Heteronuclear multiple bond correlation. The value of the 2,3 bond
Phase sensitive. Attempts to extract more long range coupling constant will be changed
correlations than hmbc over a range of coupling between two values. The default
constants. Preferred over Hmbc. is between 5 and 10 Hz. Can
discriminate between correlations
due to 2 bonds and those due to 3
bonds.
Hsqctocsy, Heteronuclear multiple bond correlation with tocsy Same as hsqc and tocsy.
Hmqctocxy coherence transfer. Provides correlations from a
carbon to several other hydrogens along a chain
(CH–CH–CH–H). Useful for interpretation of
crowded proton spectra.
Hetcor, coloc Heteronuclear correlation.
Uses direct 13C detection and it is therefore much less
sensitive than newer methods. Obsolete, do not use
unless you have a very good reason.
Inadequate Homonuclear 13C–13C correlation.
Extremely insensitive. Use only with 13C-enriched
compounds.

References
1. Derome, A., "Modern NMR Techniques for Chemistry Research". Pergamon Press
1987.
2. Claridge,T., "High-Resolution NMR Techniques in Organic Chemistry". Pergamon
Press 1999.
3. Sanders, J. K. M., and Hunter, B. K., "Modern NMR Spectroscopy. A guide for
Chemists”, 2nd Ed. Oxford Press 1993.
4. Reynolds, W., Enriquez, R. “Choosing the best pulse sequences, acquisition
parameters, postacquisition processing strategies and probes for natural product
structure elucidation by NMR spectroscopy”. J. Nat. Prod. 2002, 65, 221-244.
5. Berger, S., and Braun, S. “200 and More NMR Experiments”. Wiley-VCH, 2004
6. Varian, Inc. VNMRJ online documentation. http://www.umich.edu/~chemnmr/docs.html