Offline processing of NMR spectra on PC’s using Mestrec

Nancy Mills, Trinity University

Fall, 2002

The software

The program used for processing the data for NMR spectra, the free induction decay or FID, will be available on the NSF-NMR website, under the Department of Chemistry, Trinity University. These processing directions will also be stored there. You may also down load the processing program for free over the internet. The program was developed and is maintained by a research group at the University of Santiago de Compostela. While the program is free, they ask that you register to use the program so that they can continue to receive funding for its support and development. So, if you download the program, please follow the instructions on the website for registering. The URL is http://qobrue.usc.es/jsgroup/MestRe-C/MestRe-C.html. You are interested in MestRe-C 2.3 (for 1D spectra) so go to download and select this program. After the program is transferred to your computer, you will need to unzip it. I have found that the location that you put the unzipped program needs to be different from the location of the downloaded, zipped (compressed) version. Let me know if you have difficulties. If you have a Mac, there is a free program for it and the link is located on the web site listed above. I have an older version of directions for that software, SwaN-MR. If you are operating on a Mac and need these directions, talk with me.

The data

The FID’s will be stored on the website as compressed files. You will need a program that uncompresses (unzips) files. If you do not have one, a good, free one is BigSpeedZipper which is available for download from http://www.bigspeed.net. Follow the links in the NMRSite from participating institution, to your school, to the experiment you are doing. You will see a link to your data, by your name. Click on that link. You will probably want to save the zip file on your own computer. After you unzip the file, you will see five files, FID, LOG, PROCPAR, SAMPLEINFO and TEXT. You will probably want to create a folder to put these files in, because the same five files are created for every NMR experiment and if you are saving your files to the same computer used by another student in your class, you will write over their files.

Processing spectra.

Open the application program for MestRe-23 by selecting Run from the Start menu, if you are using a Windows machine, and browsing until you find your copy of the MestRe-23 program. In the MestRe program, until File, select open, then navigate until you get to the folder with your data. Open that folder and select FID. The first time you use the program, you will need to go to the drop down menu on the lower left corner, labeled File Type and select “Varian VXR-Unity”. Then select open from the box next to file name. The FID is now be displayed on the screen. If you get an error message at this point, you probably have not selected “Varian VXR-Unity”.

1. Under Process, select Fourier Transform and select the default settings. You may change the size of the spectrum using "+" to increase the size and "-" to decrease the size. If the phasing is bad, go to Process and select Auto-phase correction. This is usually good enough to use. Put the pointer on top of the baseline and drag it down until the baseline is about half an inch above the scale.

2. To set the reference signal, look for a peak at around 0 ppm, which might be very small. Select Tools/Reference in the menu bar. The mouse pointer will change to a new shape. Move the mouse pointer over the peaks to see the current chemical shift of each peak. Click over the desired peak to invoke the Solvent Signals Dialog Box. In the dialog box, select the corresponding chemical shift or introduce a new value. Click on the Ok button to reference the spectrum. Press the ESC key or click Cancel at any moment to exit the command. If the peak is too small to accurately position the cursor, zoom in on the spectrum, see step 6, set the reference, and then return to the full spectrum (step 6)

3. To integrate, go to tools and select integrate. You can use the mouse to create a box around each region of peaks; create the box so that it will inscribe the area you need to integrate. Use a single box for each discrete set of peaks. If you make a mistake, select escape, and click with the mouse on the integral and you should see a box. If you select delete the box and the integral will disappear. You may normalize all integrals to one peak, that is set the integral on that peak to 1 and have the other peaks' integration set relative to that, Place the cursor over the value or the trace and click once with the right mouse button; this brings up a dialog box in which you can set the value of the integral. All other integral values will be changed with respect to this one. By default, the first integral is the reference value (1.00). If the integral is too large, then select escape and place the mouse on the integral so that you get the box. Then move the mouse to a corner so that you get a diagonal arrow and use that to adjust the size of the integral.

If the integral is not level with the baseline, select tools, integral correction, apply.

When several peaks are close together, you may want to integrate the peaks together, and then do an expanded plot of the region (see step 5 below) with separate integration, because the integral values may appear on top of each other if they are too close.

4. To identify peak frequencies, go toTools, select peak picking, then peak picking again. Use the mouse to create a box that will include the peaks of interest to you and the program will automatically calculate the peak frequencies. The peak frequencies (in ppm and hertz) will appear in a separate file called results which can be selected under tools, peak picking, edit. When this box is open, you can print it using the print command. If you change your mind about the area you are peak picking in, you will need to go to the results table and delete the incorrect information already listed.

5. To give a title and information about your spectrum, select edit, add text and type in your title.

6. To examine an expanded portion of the spectrum, which will be printed on a separate page, go to view, zoom and use the mouse to draw a box around the area to be expanded. It will may then be integrated and "peak picked" as above. To leave the zoom mode, go to the menu bar and select the icon which looks like a spectrum and has “full” written below it. To calculate the coupling constant, select Tools/Measure Coupling Constants in the menu bar. The mouse pointer will change its shape to indicate the new operation mode, and the Coupling Constants Dialog Box will be invoked. Click on the peak whose coupling constants you want to measure. Once clicked, this peak will become the origin peak, and will be marked with a red arrow. The mouse pointer will change again its shape, thus indicating that you are measuring a coupling constant. Move the mouse pointer on to another peak to measure the coupling constant. Click on it to add the shift of the two peaks and the value of the coupling constant to the grid of the dialog box. Click on another peak to obtain the value of its coupling constant with the origin peak. To select another origin peak, press the Reset button in the dialog box or click the right mouse button on the new origin peak. Select print to print the list of coupling constants when the dialog box is open. Press the Close button to exit the dialog box.

7. To print, go to Print under file.

I like to select print preview, just to see what I am getting, but this takes a little more time. Print as you would normally do. To print the results screen, select results from windows, and then print it.

8. When you are finished, exit MestRe-23.

Known problems: In the integration mode, if you click with the left button on the bottom of the spectrum, you will get a red line near the bottom of the spectrum. I can’t figure out any way to get rid of it, so you will need to delete all integrals, under tools, integration, and delete all integrals.

You may want to either save the full spectrum or print it out before zooming because the processing you do on the zoomed spectra affects the full one.