IMSERC NMR MANUAL 02: Basic Processing of Varian 1D NMR Data
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1 IMSERC NMR MANUAL 02: Basic Processing of Varian 1D NMR Data Last updated: July 15, 2011 by Josh Kurutz This manual describes how to process NMR data on the offline processing computer in the IMSERC lab using VNMRJ. Access from other computers is somewhat different, and is covered elsewhere. Locating and copying your data to the working drive 1) First, you need to locate your data and transfer it to your folder on the read-writeable working drive, which can be accessed as an exterior hard drive from any other computer. Start by locating your spectra on the spectrometer computer. First, open the file browser by clicking on the Computer button on the Linux desktop 2) Next, navigate to your spectrum saved on the spectrometer computer by: a. Clicking on Filesystem to get to the topmost directory, / b. Clicking on nfs to get to the network file sharing folder c. Clicking on the link to the spectrometer on which you have your data, followed by clicking on the links necessary to get to the folder where you saved your data 1
2 3) Now that you can see your data folders, you need to find your destination folder on the working drive. Locate it by: a. Clicking the icon on the Linux desktop labeled working on imsercfile.chem.northwestern.edu b. Clicking on your group s folder, then clicking on our own folder. If you d like to create a new folder, simply right-click and select create new folder or go to main menu and pull down to File New folder 2
3 4) Next, simply drag your data folders over to the destination on working : Now you re all set. Actually, you CAN read spectra directly from the spectrometer computers from the lab s offline processing computer, BUT you don t have privileges to write to that folder, so you can t save peaklists, integrals, processing parameters, etc., and you cannot print to a file. In practice, it will probably be better if you re in the habit of copying your data to the working drive. This will alo make it easier for you to access your data remotely. 3
4 Opening your spectrum in VNMRJ 5) Now you need to start up VNMRJ and open your data. Start by clicking the VNMRJ icon on the Linux desktop, unless the program is already running. 6) Open your file in VNMRJ by either clicking the open file icon,, OR by pulling down File open from the menu bar. You ll see a bunch of files from the last directory that had been opened. In this case, you can see from pulling down the Look In tab that the last person to use this computer s VNMRJ took a spectrum located on the P500 spectrometer: 4
5 7) From here, change your directory to: /nfs/working/(your group folder)/(your folder)/(your data folder) 8) To open your spectrum, either double-click on the desired.fid folder, or highlight it and click the Open button. Your spectrum should appear in the spectrum display window: 5
6 Adjusting Vertical Scale Now that your spectrum is on display, let s quickly review basic zooming in/zooming out and intensity adjustments. 9) Let s start with intensity adjustment, also known as vertical scale adjustment. First, centerclick above the baseline to increase the vertical scale of the spectrum: 10) To restore the vertical scale such that that tallest peak fits within the viewable area, click the Autoscale button in the Process Default panel. 6
7 Cursor manipulation, Zooming in, Zooming out 11) Examine some of the prominent lines in your spectrum more closely by zooming in on them. First, place your left cursor to the left of the peak with your left mouse button : Next, place your right cursor to the right of the peaks by right-clicking : Next, click the zoom in button (magnifying glass with + sign, ): 7
8 Phasing Though the spectrum you see immediately after acquisition usually looks presentable, the software has already automatically made several adjustments to make it look good. One of these adjustments is phasing. We won t go into the digital sampling theory behind this procedure, but we will cover the aspects concerning spectrum appearance. Phase adjustment in VNMRJ is handled differently than in other programs. 12) Here s what the spectrum looks like when the phase is slightly off. The key test is drawing an imaginary line through the baseline on one side of the peak and determining if it connects to the baseline on the other side of the peak. You can see from the dashed red lines here that these peaks fail that test: 13) Before we cover manual phase adjustment, let s consider automatic phase adjustment. Most of the time, it will be sufficient to autophase your data using the autophase button in the Process Default panel: 8
9 14) To make manual phase adjustments, you need to enter the phasing mode of the software by clicking on the phase mode button at the side of the VNMRJ window :. You won t see anything change in the spectrum display yet. You should start adjusting the phase on the right-hand side of the spectrum, get the peaks on the right-hand side of the spectrum looking good, then move on and adjust the peaks at the left-hand side of the spectrum. You may need to go back and forth between right- and left-hand sides of the spectrum before the whole spectrum looks well-phased. To start, left-click on a region toward the right-hand side of the spectrum; here, I clicked near 2 ppm: 15) Next, left-click-and-hold-the-button-down and drag the mouse up and down. You ll see the appearance of the spectrum change, for better or worse. In this case, I overadjusted the phase, so the baselines on either side of the peaks tilt in the other direction: 9
10 16) Continue adjusting the phase until you get the peaks at the rightmost side of the spectrum properly phased. For fine adjustment, right-click-and-hold and move the mouse up and down. Do not worry about the peaks at the left-hand side of the spectrum yet. 17) Now adjust the phasing of the left-hand side of the spectrum. Start by left-clicking toward the left-hand side of the spectrum. Here, I clicked near 9.3 ppm and got a phasing window from approximately 7.8 to 11.0 ppm: 18) Next, adjust the phase of the leftmost peaks by left-click-and-holding the left mouse button and dragging it up and down. Stop when the leftmost peak is phased correctly; note that you may need to go back and adjust the phase of the rightmost peaks again, as shown here: 10
11 19) Proceed to go back and forth, phasing the two sides of the spectrum until the whole spectrum is phased correctly, as shown here: 11
12 Baseline Correction 20) If you adjust the vertical scale on your spectrum, you may find that the baseline iscurved, tilted, or offset from zero intensity. If you integrate your peaks at this stage, the intensities will be inaccurate. To correct the baseline, you need to tell the software which regions of your spectrum represent signals and which represent noise, then ask the software to perform a baseline correction. The software fits the noise regions of the spectrum to a spline function, then subtracts that function from your spectrum. The software distinguishes signa; from noise regions using the integration feature. Here is a close-up view of a spectrum with a baseline that needs some correction. Note that the spectrum must be properly phased before undergoing baseline correction. If your spectrum is simple, you may be able to correct your baseline with two mouse-clicks. First, in the Process Default panel, click the Find Integrals button. Second, click the BC correct button just below the Find Integrals button. With luck, this procedure will correct your baseline adequately: 12
13 If you have broad or low-intensity peaks, the automatic find integrals button may not identify your peaks well, and you will have to identify your integral regions manually. Please see the next section on integrating, then click the BC Correct button after you ve defined your integrals. Integration 21) There are many ways to approach manual integration, but here is one that we find to be robust. First, while your spectrum is being displayed with the vertical toolbar on the left of the screen, click on the show integrals button: This will draw green integral lines indicating intensity over your peaks, but no green lines will be drawn over regions of noise. If you click on that button a second time, green lines will be drawn over the regions of noise as well as your peaks. Clicking one more time will remove the green lines altogether. Keep in mind that when the green integral lines are displayed, your vertical scaling function (center-click) only affects the integral display. If you wish to adjust the intensity of your spectrum, you have to click the above button until no integrals are displayed. If you like the integrals that the software picked automatically, proceed to the integral normalization task. To manually adjust your integrals, you may be best off by clearing the integrals by clicking the Clear Integrals button in the Process Integration panel; you ll see one continuous integral line proceeding from left to right, increasing in height every time it encounters signal intensity: 13
14 Next, you need to add boundary points points that mark the left and right boundaries of an integral region. VNMRJ calls these reset points. Establishing a reset point toggles the identity of the integral line to the right of it. To start editing your reset points, 1) Click on the Interactive resets button in the Process Integration panel, or click on the integral-line-with-orange-handled-scissors button in the spectrum s vertical toolbar,. 2) Locate the peak farthest to the left of your spectrum (, ), then left-click on a point to the left side of that peak, where you wish to define the start of that integral. You ll see the integral line to the left of this point become dashed and the integral line to the right remain solid. The dashed line identifies a region of noise, and the solid line identifies a region to be integrated. Next, left-click on a point to the right of the peak, where you wish to define the end of that integral. You ll see that the integral line to the right of this peak becomes dashed, and the line to the left remains solid, defining the region between your reset points as a peak integral region: 14
15 Continue in this fashion, going from left to right across your spectrum, defining left- and right-boundary points for each integral region: Note that you do not have to integrate every peak in your spectrum. However, if you are using these integral regions for baseline correction, you should integrate every region with significant intensity, then apply baseline correction. After baseline correction, you are free to remove integral reset points for analysis. To REMOVE RESET POINTS, simply hover your cursor near a rest point and right-click,. Note that this toggles the identities of all regions to the right of that point, turning integral regions into noise regions and vice versa. If you are removing a single integral region, be sure to remove both of its boundary points. Note that the right-click function always removes the closest reset point. So if you click once you remove the nearest point, and even if you do not move your mouse, right-clicking again will remove the next-nearest reset point. Right-clicking rapidly for awhile will delete ALL of your reset points! After you have applied baseline correction and defined the integral regions of interest, you will probably wish to normalize your integral values to a single peak. Identify a peak for which you know the number of corresponding 1 H atoms. Then, 1) Click on the integral region of that peak to put a cursor there. 2) Click the (Normalize area to) Single Peak button in the Process Integration panel. 3) Define the value for the integral by typing in the box shown and hitting <return>. 4) Click the Set Integral Value button. 15
16 Now, to display the calibrated integral values, click the Show Normalized Values button. The values will be displayed both beneath the spectrum scale and in the table window of the Process Integration panel: Peak-picking 22) To determine the precise position of your peaks, you should proceed with peak-picking. To do this, you first obtain a an interactive threshold cursor (an orange horizontal bar) and place it below the tops of the peaks for which you want frequencies, but above the peaks for which you do not want frequencies. Once that threshold is placed, you click the Find Peaks button to obtain peak frequencies. To obtain the orange threshold cursor, 1) click on either the threshold button in the vertical spectrum toolbar at the left of the spectrum display, or click on the Peak Threshold button in the Process Default panel. 2) One the threshold is on the screen, leftclick-and-hold the mouse and drag it up and down to set its position: 16
17 After the threshold is set where you want it, then click the Find Peaks button to display peak frequencies: 23) 17
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