Reprocessing NMR Spectra in ACD Spectrus Processor

IntroductionNMR spectroscopy is one of the most powerful analytical tools available to synthetic chemists and has become the first choice tool for structural analysis of a compound or to examine a reaction mixture, especially in organic chemistry where most molecules are likely to be diamagnetic and consist predominantly of I = ½ 13C and 1H atoms which yield the key structural information.

However, despite advances in modern computing, it is still rare for an NMR spectrum to be useful to its full extent in the automatically processed version that is initially provided in printed form from a spectrometer. It is therefore usual, if not essential, to reprocess recorded spectra in order to make the most of the data acquired.

Opening files

On the left hand side is the file browser to allow you to select NMR files you have downloaded. Navigate to the location of the sample NMR files you have already downloaded, then open up the Ethylbromoacetate folder and within that open the Proton-CDCl3 folder. In this folder locate the FID, short for ‘Free Induction Delay’. Click on this file, then click and drag the file to the right hand pane in order to open the file.

Viewing spectrum parametersBefore spending time processing a spectrum, it is often a good idea to check the ‘spectrum parameters’ which lists the metadata associated with the spectrum. This is a mixture of data automatically generated by the spectrometer and information input by the NMR operator when the samples were loaded. The most useful section is the comment field which allows you to check that it is the spectrum you were expecting. This field usually contains the name of the sample owner and the title they gave to the sample. The spectrum parameters can be viewed by clicking View | Panels | Spectrum parameters. This will display a box (you may wish to resize it) listing all the parameters. Check you can find the ‘Comments’, ‘Nucleus’, ‘Solvent’ and ‘Frequency’ fields.

Fourier transformA FID is recorded by the NMR instrument and is the resonant signal(s) decaying after the excitation pulse, over time. The first thing that is usually required when opening a FID is to perform a ‘Fourier Transform’ (FT) to convert the signal from the time domain into the frequency domain. Spectrus performs this function automatically, but it can be useful sometimes to turn this off and view the raw FID which should have a decaying signal from left to right. In order to temporarily turn off the FT, in the bottom left of the screen there is a ribbon of functions. The first in ‘Process’ is the ‘Fourier Transform Options’ menu. Unclick ‘Apply FT’ to show the raw FID. Once you have seen this, reapply the FT and exit the menu.

Zooming inAlong the toolbar are a set of tools for zooming in onto the spectrum. The first icon is the horizontal zoom.

Click to the left of the chloroform peak (around 7.5 ppm) and drag across to the right until you have reached around 1 ppm and release the mouse.

The top pane on the right should show the zoomed spectrum. Below is an overview of the whole spectrum which can be useful if you are looking at a complex spectrum. At the bottom is the ‘spectral data viewer’. Whilst these can be useful, it can be worth turning these panes off to gain

more screen space. View | Zoom area turns off the overview spectrum. The Spectral Data table can be hidden by clicking this button:

The spectrum should now be filling the screen.

Other zoom options

The second icon in the zoom toolbar is the rectangular zoom. Click on this and draw a rectangular box around the peaks between 4.0 and 4.5 ppm. The magnifying glass with the green arrow reverts to the previous zoom area (Zoom undo) and the magnifying glass with a minus zooms out to show the whole spectrum (Show whole spectrum). Once you are happy with these tools, leave the spectrum zoomed in like the figure above.

PhasingSometimes the phasing of the spectrum can be non-optimal, resulting in poorly displayed peaks. In this case the phasing is good, but it is worth experimenting with the phasing to see what effects this tool can have. Click on the ‘Fourier Transform Options’ icon, then choose ‘Phase Correction Options…’ A box will appear (you may now need to drag this box so that you can see the toolbar still). Click on ‘Mouse Phasing’ then click around a peak and drag up/down with your mouse and see the changes in the phasing. Mouse phasing works by applying corrections to the spectrum (you need not worry about the details at this stage). If an area is poorly phased, then you move the mouse to the area that is worst, then click and drag the spectrum to rephase it. A poorly phased spectrum is displayed below.

Mouse phasing remains selected until you reclick on the Mouse Phasing button and this will change it from blue to grey. In order to return to the original phasing, either click the undo button:

Or click ‘Auto Phasing’ to rephase the spectrum automatically again.

ReferencingThe next stage in NMR processing is to set a signal to a known PPM. For NMR this is carried out either by setting the residual protic solvent in the deuterated solvent to its known value, or by reference to a known additive (for example TMS). For this example, CDCl3 is the solvent, and this is what we will reference the NMR to. A number of very useful papers have been published in journals listing the ppm shift for common solvents, for example Fulmer et al. http://dx.doi.org/10.1021/om100106e

These papers can also be very useful for identifying contaminants in NMR spectra, which may typically include the solvents used in the preparation (for example the solvent used in a recrystallisation).

In order to reference the chloroform peak in Spectrus, click the reference button on the bottom ribbon.

The mouse pointer will change to having ‘ref’ displayed next to it. Click on the signal due to the chloroform (in this case furthest left). A new window will open. Choose Chloroform-d in the list and click ok. This will set Chloroform to 7.27 ppm. If you wished to set this to a different value, you could enter a number into the shift value in the box (right). Click OK to apply the referencing.

IntegrationIntegration is one of the most useful reasons for reprocessing NMR spectra. Integration allows you to confirm that the number of protons giving rise to each environment is as expected for a molecule, or more usefully in mixtures of compounds, integration allows you to calculate the ratio of each compound in the mixture.

Whilst Spectrus has automatic modes for integration and peak detection, performing these tasks manually gives you more control and it is important to be able to manually perform these tasks. Manual integration can be particularly important when trying to quantify different components in a mixture by NMR, for example in a reaction where two stereoisomers are formed.

By default, the manual integration tools are hidden in Spectrus. To show the toolbar click the arrow on the right hand side of the ‘Peak Detection’ ribbon and choose basic workflow. This should alter the ribbon to have the following icons.

To integrate this spectrum, click the integration button on the ‘Peak Detection’ toolbar. On the next toolbar, manual should already be selected, but if it hasn’t been selected, click on manual. To integrate a peak, click on the left of one peak and drag across the peak until you are to the right of the peak, then release the mouse button.

This will integrate the peak, but it is unlikely to be the correct value for the number of protons giving rise to that signal. For the peak selected above (the CH2 of the ethyl group), this would be 2. Right click on the integral value and select ‘Edit Integral Reference’. Change the value to 2 and click ok to apply the change.

Then use the mouse to integrate the remaining two signals in the spectrum (these should be ~2 for the CH2Br signal, and ~3 for the CH3. If you wish to delete an integration, right click on an integral and choose ‘Delete Integral’. Once you have fully integrated your spectrum you will need to reclick on the ‘Manual Integrals’ button in order to deselect the button (it will change from yellow to blue).

Peak pickingPeak picking, as well as helping identify the signals and enabling reporting, also allows coupling constants to be calculated. There are two options available for peak picking: by level or one by one. The first option is peak level, which can be a very useful and quick way of peak picking (often in conjunction with later refinement using peak by peak picking). Select the ‘Peak Analysis: by peak level’ tool.

The mouse will change to having a red crosshair and lines following it. The horizontal line is the important one, peaks above the line will be picked and those below will not. Try experimenting with different levels. The other option is peak by peak picking using the ‘Manual Peak’ tool.

Select this option, then click on an individual peak to label it. Click again to remove the label. Once you are happy with your peak picking, click the green tick to accept the peak picking. You should have something similar to the below.

Coupling constants can be calculated using the difference between the peak splittings (in ppm) and multiplying by the spectrometer frequency (in this case 300 MHz) to obtain a coupling constant in Hz. For example in the above spectrum, the quartet at ~4.25 has peak pickings of 4.28, 4.26, 4.23, 4.21. Taking the difference between these peaks (eg 4.26-4.23 = 0.03 ppm), then multiplying by the spectrometer frequency (0.03 ppm × 300 MHz) gives a value of 9 Hz.

ExportingAt this stage, you should have a processed NMR spectra, although further annotations are desirable, it is often easier to carry these out once the PDF has been created. To export the spectrum as a PDF, click File | Export report to PDF | standard.

The first time this is done, it is necessary to change what options are included on export. On the spectrum tab, select ‘always landscape orientation’; ppm for units; ‘normalised intensity’ for display mode and Zoom Region (unclick Whole Spectrum). For tables, uncheck everything. In view, uncheck everything except spectrum labels, integrals curves, integrals values, vertical scale and horizontal scale. On the text tab, check only page title and enter something meaningful. It is generally a good idea to include the nuclei, frequency, compound name and solvent (eg 1H NMR spectrum (300 MHz) of Ethyl bromacetate in Chloroform-d). You may additionally wish to include the spectra reference (eg date and number), the ‘document name’ box is a good place to include this.

Once you are happy with the information entered, click ok and you will be asked to name and save the file. Open the PDF to check you are happy with this output.

It is also worth saving this processed spectrum. Choose, File | Save. The spectrum will be saved as an .esp file.

Annotating the PDFsIt is normal to add a structure and assignment to a processed NMR as part of your evidence for having made a compound. Whilst this can be done within ACD NMR, it is often easier to perform this step by editing the PDF in a PDF editor. On classroom PCs Adobe Reader (the default programme PDFs are opened in) performs this task well.

The first step is to create a ChemDraw structure. This is not covered in this guide (you should have already completed the ChemDraw learning package). Construct a structure similar to the below and label each environment. In this case, the structure has been labelled to reflect the naming used in the NMR interpretation guide. How you label a structure is up to you, but it should be logical.

You can then select and copy the structure from ChemDraw, and paste it directly into Adobe Reader (you may need to resize the image after pasting). Sometimes the structures get distorted upon pasting, if this happens, paste it first into a graphics programme (eg Paint), then recopy the image from there.

Text annotations (for example to label each individual peak) can be added by selecting ‘comment’ on the right hand side, then choosing the ‘add text comment’ tool (icon is a ‘T’). Click where you wish to write on the spectra and type. When you have annotated your pdf, choose file | save as. It will ask you to confirm that you wish to overwrite the existing file. You should now have something similar to the below.

Compare your spectrum to the PDF in the processed folder that you downloaded earlier. You may now wish to try processing some other examples in the folder. For each example, there is a processed PDF and a processed .esp file which can be opened in ACD NMR.

Reporting dataChemists use a very condensed, structured style to report NMR data as part of an experimental procedure. These shorthand formats were originally developed to use a minimal amount of space within print journals. Whilst this is less of a limitation today, chemists continue to use a very shorthand format.

Differing stylesWhilst all journals share similarities in the way their data is reported, different publications use differing house styles. Detailed guidance on each style can usually be found on the publishers’ websites under ‘Author guidelines’ and these will detail their individual requirements. When writing up NMR data you may be directed to use a particular style, or you may be allowed to choose your own style. In either case, it is important that your data is presented in a consistent format. The easiest way to become familiar with the different formats is through reading journals, for example comparing your data to those reported in the literature.

Proton NMRFor each data set, the spectrometer frequency and solvent should be reported, then for each proton environment:

Chemical shift (or range for a multiplet) Number of protons giving rise to the signal Multiplicity (singlet, doublet, triplet, quartet or multiplet etc) Coupling constants (in Hz) Assignment

Carbon NMRFor each data set, the spectrometer frequency and solvent should be reported, then for each carbon environment:

Chemical shift Assignment Where coupling is seen (eg compounds containing 19F) then multiplicity and coupling

constants should be reported

Other nucleiAny other NMR nuclei should be reported in a consistent fashion relevant to what is being recorded. This might include 31P, 19F or 11B for example.

ExamplesEthyl bromoacetate1H NMR: (300 MHz; chloroform-d): 4.25 (2 H, q, J 6 Hz, CH2), 3.83 (2 H, s, CH2Br), 1.31 (3 H, t, J 6 Hz, CH3).13C NMR: (75 MHz; chloroform-d): 167.2 (C=O), 62.3 (CH2), 25.9 (CH2Br), 14.0 (CH3).

Ethyl bromoacetate

1H NMR: (300 MHz; chloroform-d): 4.25 (2 H, q, J 6 Hz, H-3), 3.83 (2 H, s, H-1), 1.31 (3 H, t, J 6 Hz, H-4).13C NMR: (75 MHz; chloroform-d): 167.2 (C-2), 62.3 (C-3), 25.9 (C-1), 14.0 (C-4).