Basic processing of 1D NMR spectra in MestReNova

Transcription

1 Basic processing of 1D NMR spectra in MestReNova This brief tutorial will introduce you to the basic operation of the MestReNova (MNova) NMR processing software. For this example lesson, we will be working with the spectrum for ethyl acetoacetate. Using this tutorial you will learn the following: 1. How to download and open an NMR file (a.fid file) within MNova 2. How to view portions of a spectrum, including zooming in to signals and zooming out to view the full spectrum 3. How to peak pick to list a peak frequency in units of Hz or PPM. 4. How to integrate the area under a signal and change the base integral value 5. How to annotate, save, and print your processed spectrum as a PDF.

2 Starting MNova Begin by launching the MestReNova (MNova) software program (double click the desktop icon or launch it from the Start menu). The software will take a few moments to load. Ignore any license warnings or ask your TA if you are having difficulties. 1. Opening FID files After starting MNova, you also will need to download the FID file for the example molecule ethyl acetoacetate. You can find the ethyl acetoacetate fid on the 347 class website by navigating your browser to the website and clicking through Software & Web Resources > NMR Software. You will see a link here for the ethyl acetoacetate.fid.zip file. Click on the link and it will save it automatically to the Downloads directory on the computer. Now you will need to open the FID file using MNova. This FID file is the file which contains the raw data acquired during the NMR experiment. Navigate to the File menu, select Open, and locate the FID file you downloaded. It should be located in the Downloads directory. You can open the zip file directly, without needing to extract it.

3 After opening the FID in MNova, you should now see the full spectrum in the main window. MestReNova has automatically applied a Fourier transform operation on the raw data to arrive at the spectrum you are viewing. Next, we will see how to zoom in and explore different signals within the spectrum.

4 2. Exploring the NMR Spectrum Now you can explore the various parts of the spectrum using some of the tools within MNova. There are a number of useful tools on the top tool bar. 2.1 Zoom in The magnifying glass with the (+) icon above it is called the zoom in tool, which will allow you to select a region of the spectrum and zoom into that area. Use it by clicking the mouse button down and dragging it across an area to zoom in as shown below: Click and drag the zoom in tool around a signal Release the mouse button to zoom in on that area.

5 2.2 View full spectrum To return to the full spectrum view, select the Full Spectrum button on the toolbar. This will display the entire spectrum again, and you can then zoom in close on a different signal.

6 3. Peak analysis In order to analyze a spectrum, it is necessary to obtain several pieces of information by examining individual signals and their associated peaks. Some of the most basic information includes: The chemical shift of a peak (in ppm and/or Hz) The splitting pattern of a signal (triplet, doublet, quartet etc.) The separation between two peaks within a split signal (in Hz) To obtain this information within MNova, we will be using a Peak Picking tool called Peak by Peak analysis. Start by clicking the Peak picking tool drop down arrow as the red circle above shows, and then select Peak by Peak It is very important that you use the Peak by Peak tool and not the Automatic option, otherwise MNova will try to figure things out on its own and make mistakes, as well as make it more difficult to find the information you need. Now that you have the peak picking tool selected, you can pick a particular peak and click with the mouse button. Doing so will display the peak frequency (in units of ppm) on top of the spectrum for you to see.

7 Try this: 1. Display the full spectrum 2. Zoom in to the quartet that is between 4.15 and You should see something like the image on the right. 3. Now choose the peak by peak tool from the Peak picking drop down as shown above and pick the four peaks along the signal. 4. If you look at the top of the window you will now see the four peak signals listed in ppm (4.20, 4.19, 4.18, 4.16). It should now look something like this: Notice also you can see the value (in ppm) along the x axis at the bottom of the spectrum. It would be more useful now to see the values of the peaks in units of Hz, so you can calculate the coupling constant J for this signal. We have to tell MNova to display peaks in Hz rather than ppm.

8 Displaying peaks in Hz units If you use the right mouse button and click on the white space above in the spectrum, a small context window will appear. Choose properties Right click in the whitespace of the spectrum Choose Properties You will see a list of items for which you can change the properties. Select Peaks

9 Now look on the right side of the window and find the Units drop down box (which will say ppm). If you click on the drop down box you will see the option to use Hz : Select Hz, and hit the Apply button. Finally close this window by pressing the Ok button. Alright, now you should see your peaks listed in units of Hz at the top of the spectrum. It will stay this way for all the peaks you pick within the spectrum until you exit the program or change the units back to ppm using the properties menu.

10 Now you can view the full spectrum and use the Peak by Peak tool to assign Hz values for all of your peaks within the spectrum. You will need these values in order to assign coupling constants to the signals within the spectrum. Once you have completed this, move on to the next section to learn how to integrate each of the signals.

11 4. Peak Integration The area under the peaks within a signal can be determined using the integration tool within MNova. We will be using the manual integration tool which will allow us to measure the ratios of protons within a given signal. Click the drop down arrow next to the integration tool button and select Manual You can now click and drag an area around a signal and an integral will be calculated for that signal.

12 You will now see a new integral curve above your signal and the normalized integral value below the signal. It is important to note that THE FIRST INTEGRAL YOU DRAW WILL AUTOMATICALLY ASSIGNED THE VALUE 1. All subsequent integrals you draw will be some factor of this first integral. You will learn how to change this default value in the next step. Continue by integrating each of the signals in your spectrum. When you are done, you should see something like this:

13 The values for your integrals might be slightly different, which is fine. Now suppose you know that the integral for the quartet at ~4.2 ppm not actually 1.00, but in fact more like This is easy to change. You can edit the value for any single integral you wish and the software will automatically adjust all the remaining integrals accordingly (their ratios were determined when you drew the actual integration curve). All of this is done using the integral manager.

14 How to access the integral manager: Select the manual integration tool as before Move the cursor over the integral span line below the signal until it is illuminated as shown Double click the mouse button You can also just double click the integral curve symbol above the peak signals as well

15 This is the integral manager dialog box. Here you can adjust the normalized value of the selected integral by typing in a new value (such as 2.00) and pressing the Enter button. When you do that, it will adjust this integral, and all the other integrals in the spectrum. You can use the left and right blue arrow buttons at the top of this window to navigate through your integrals. Do not adjust any of the other values or press the various buttons, as it will change your integrals to values you do not expect. Next you will learn how to save your processed spectrum and export it as a PDF file for your lab notebook records.

16 5. Annotating, saving, and exporting your work. You can save the processed MNova file by using the file menu and choosing save as, just as you normally would in any other software program. The file format is a.mnova file, and it will contain all of the formatting and processing you have performed on the file. You can yourself this file, save it to a cloud file service/dropbox, etc. To annotate (make notes) on your spectrum, such as the spectrometer frequency, your name, the experiment number, or signal assignment notes you can use the text tool. Find this tool down in the lower left hand corner of the screen where the arrow is located and click the drop down menu. Select Text. You can use this to enter text anywhere on the spectrum document. Font size and color options are right along the bottom portion of the window.

17 Exporting as a PDF Before you exit, you should also make sure to save the file as a PDF which will print nicely and can be viewed on any computer without the need for MNova. First, use the zoom in tool to expand the region that you want visible in the PDF file. When you are happy with the appearance of the spectrum choose Export to PDF Type in a file name and click save. You now will find a PDF of your processed spectrum which you should save to a permanent location for the class and also print a copy for yourself to have in your lab notebook.