NMR Spectral Processing with MestReNova (MNova)
1. Download the MNova file posted on Canvas.
2. Open MNova. Ensure the correct interface theme so that these instructions make
sense. Set the interface theme to Modern by going to the MestReNova menu at
the top and selecting File, then Preferences, then in the Interface tab, in the Theme
setting, select Modern, and click OK.
3. Open the downloaded MNova file. Once open, MNova should look similar to this:
You can increase
or decrease the y-axis using
the controls on the right
sidebar. You can zoom in
using the
zoom tool, and you can restore the
original view using the full view tool on the right
sidebar. Try zooming in on the
aromatic region and notice how the figure below
fills the window up with the
peaks.
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4. Chemical shift referencing: newly acquired spectra have chemical shift values that
are approximate, but every spectrum needs to be manually referenced in order to
ensure accurate chemical shift values. The solvent in which your compound is
dissolved comprises mostly (~99.9%) deuterium atoms instead of hydrogen atoms.
That is because deuterium (2H) is invisible to the NMR spectrometer at the usual
frequency range for a 1H spectrum. Therefore, you don’t see a huge solvent peak
due to the huge concentration of solvent, which would drown out the desired solute
signals. Nonetheless, there is always a small amount of residual protonated
solvent, which does give a signal in the 1H NMR spectrum. We know the exact
chemical shifts of the common solvents, and therefore we can use the signal from
residual protonated solvent as a standard to which to reference the chemical shift
of all of the other signals. To do this step correctly, you will need to know which
solvent was used to take the 1H NMR spectrum of the compound given. In the
spectrum shown below, the sample is dissolved in deuterated water (deuterium
oxide, D2O), for which the residual protonated solvent has a chemical shift at
exactly 4.79 ppm. To zoom in on the peak you want to reference, go back to full
view, and then zoom in on the region where the signal is located. Select the
analysis tab, and then press the Reference button at the left of the top bar.
Place the cursor at the center of the peak and click on the peak. A new window will appear.
In the new shift text box, enter the chemical shift for the solvent (in the case of water, 4.79
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ppm) and click OK. Now, all of your chemical shift values are accurate. Generally, you
want to have the peaks as large as possible for this step, but no larger than the window.
Note: the spectrum provided to you was taken in deuterated chloroform (CDCl3, reference
7.26 ppm).
Sometimes, if signals corresponding to the molecule of interest overlap with the solvent
signal of the sample, another approach needs to be taken to properly reference the
spectrum. Usually, NMR solvents are spiked with a molecule called tetramethyl silane
(TMS), which has one singlet that is set at 0.00 ppm. This singlet is oftentimes used to
reference NMR spectra if needed.
5. Integration: integrating the peaks will help you analyze the spectrum by
determining the relative number of hydrogens represented by each signal, and it
can help you identify signals that don’t belong to your compound because the
integrations are either way too high, way too low, or clearly a non-integer value.
Integration can also help you characterize mixtures of products.
In the Analysis tab, select manual integration. Place the cursor on one side of the
peak you wish to integrate where the peak meets the baseline. Click and drag the
cursor to the other side of the peak where it meets the baseline.
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A step curve appears above the peak; the height of this curve is proportional to the
area under the curve. In addition, an arbitrary integration value of 1.00 appears under
the very first peak integrated. All subsequent integration values are relative to this one.
This value of 1.00 may need to be adjusted to more accurately reflect the number of
hydrogens that this signal represents, but you may not yet know how many hydrogens
that peak is supposed to represent. Go ahead and integrate all the other peaks in the
spectrum. Sometimes, peaks blend together, and you need to integrate the whole
bunch; sometimes you can eyeball the break between two closely proximal peaks. It
is important that you zoom in far enough to be able to carefully place the left and right
boundaries of each peak integration. See example below.
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Once you can assign one peak or a group of peaks, you should adjust your integral
values to more accurately reflect the number of hydrogens and thereby help with your
assignments. To do so, right-click on the step curve above the peak or on the integral
value below the peak, and select edit integral to access the integral manager window.
In the normalized text field, enter an integer value for the number of hydrogens
assigned to that peak. Press enter, then close the window. All other integration values
will be adjusted automatically based on the integration value you set, and therefore
you should only need to do this adjustment for one peak. Try to integrate peaks
belonging to your compounds, not solvents.
Peak picking. MNova can help identify the exact chemical shifts of all peaks. Zoom in
on a region of the spectrum so that you can clearly see the peaks that comprise the
multiplets. Then go to the Analysis tab, and press the Peak by Peak button to manually
select all of the peaks in that region. You need to set the precision of the chemical
shift values to three decimal places (thousandths). To do so, right click on the desktop
and select Properties. In the Peaks tab, enter a value of 3 for Decimals. Here is the
aromatic region fully picked and in the correct format:
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Making Spectral Figures
1. You should always include a figure that shows all the peaks in the spectrum, with
an extra 1 ppm of flat baseline on either side. That is the full spectrum. Figures
with zoomed in spectra are helpful for visualizing detail and should also be created.
For all spectral figures, the peaks should be scaled vertically such that the tallest
peak is close to the top of the spectrum without going off scale. It is common,
however, to allow solvent peaks and other known contaminants to go off scale
vertically.
2. Solvents and peaks from unknown impurities (especially if the peaks are small) do
not need to be integrated and may be ignored.
3. Much of the credit for an NMR assignment will depend on your accurate
assignment of the signals in the spectrum to their corresponding hydrogens on the
structural formula. A properly formatted spectral figure should, therefore, include
the structural formula of the compound with letters next to magnetically
nonequivalent groups of hydrogens on the structure. The letters should also be
added next to the peaks that correspond to those hydrogens.
The best way to create a properly formatted structural formula is with Chemdraw.
After you have made the structure, label the magnetically nonequivalent groups of
hydrogens with lower-case letters using Chemdraw, and then export this file
(File/menu/export) as a png file with resolution 300 and a transparent background,
saving the file to your desktop. To insert your Chemdraw graphic into your MNova
spectrum, simply drag the icon for your saved .png file into the MNova window
with your spectrum. Click on the structure, and a set of green squares will appear
around it. The graphic should be clearly readable without overlapping with other
features of your figure.
Assign the peaks in your spectrum with letters corresponding to the hydrogens on
your Chemdraw graphic. Generate a new textbox by going to the menu Edit >
Annotate > Text; then click on the figure, type a letter, and click elsewhere to create
the object.
4. For Mac: Using the menu option File > Export to PDF, export each fully formatted
and assigned figure from MNova as a PDF file. Be sure to only export “current
page” if prompted with this question. For Windows: Choose menu option
File > Save As, then choose PNG format.
5. On a Mac: In an MS Word file, import your spectral figure by selecting the menu
Insert > Pictures > Picture from File, and then browsing to select your image file.
Add a figure caption below the figure that gives the figure number and a brief
description of the figure contents, for example, “Figure 1. Aromatic region of the 1H
NMR spectrum of ethyl acetate in CDCl3.
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6. Below are some good examples of figures of NMR spectra.
Figure 1. 1H NMR full spectrum of p-aminoacetanilide in DMSO-d6.
Figure 2. Aromatic region of the 1H NMR spectrum of p-aminoacetanilide in DMSO-d6.
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