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Tutorial 07 -- Spectrum
Spectrum is window used for viewing mass spectra. Its primary function in MSC is the evaluation of spectra and the comparison of an observed mass spectrum with the results from the autofragmentation of a structure.
1 Select the main menu option Spectrum | Show Spectrum or select the corresponding speedbutton from the Toolbar. You can also press the shortcut key, F7.
2 A new window labeled Spectrum will open. Click on the Open File button to select a spectrum file to view. Spectrum files in MSC are mass/intensity lists maintained in ASCII files with a default extension of .asc. A file named '4Clbiphenyl.asc' was included with MSC and should be present in your program Demo Files directory; select it and click OK to open it into Spectrum.
3 The mass spectrum of 4-chlorobiphenyl will be displayed. The Spectrum toolbar at the top includes Speedbuttons to Open a file, and Print the spectrum. The button with a pencil will redraw the image while the red X will clear the image and close the file. Click on the button with the (+) magnifying glass four times to increase the scale expansion to about 12% (full scale). Each click will increase the expansion. Clicking the (-) magnifying glass button will reduce the expansion of the display with each click. Clicking on the Home icon will restore the default (full-scale) conditions.
4 While holding the Shift key, point to an unlabeled peak and click the left mouse button. The mass should be labeled. Repeat the same process again but without holding the Shift key. In this case the Elemental Formula utility will pop up with the mass already entered and one of two default selections. If no structure is present then only Elements C and H will be selected and the number of formulas possible is shown. You can adjust the options further as needed. Closing the Elemental Formula window and clicking on another peak will repeat the process.
If a structure is present in the Drawing window of MSC then the elemental composition of that structure will be used to pre-define the maximum possible number for each element. To indicate this condition, a red label reading Constrained will appear on the window just to the right of the Found box. Of course, these values can be altered and other options selected as before.
5. As the mouse is moved around the image, the current m/z value is constantly displayed in a small box at the bottom of Spectrum. You can use that value to make certain you are clicking on the correct peak. Right-click on the 152 fragment peak and while holding the right-mouse button down, drag to the 188 molecular ion. When you release the button a small Neutral Loss box will open listing the two selected masses, their difference as a neutral loss, and a list of any common neutrals for that mass. In this case, the likely loss of HCl will be included.
This can be repeated as desired. Right click on the 159 fragment and draw a link to the molecular ion. In this case a difference of 29 is indicated with three possible neutrals. To clear the screen and restore the spectrum click on redraw (the blue pencil).
6. Click on Evaluate | Simple Interpretation. A text box will open with a brief, overall evaluation of the current spectrum. Click on the Close button to close the text box.
7 Select the Edit menu and choose Spectrum Label. A default label will appear on the spectrum. You can drag through the label to highlight it, and type a replacement name or other identifying information. Press the <Ctrl> key and click on the label. While continuing to hold the mouse button down, drag the label to a new location, and then release the button.
8 Select File | Exit or click the Close speedbutton (with a closed door icon on it) to close Spectum and return to the main window. Select File | Open from the main menu, and load the structure 4Clbiphenyl.msd which was included with the program. It should be in your default program Demo Files directory.
9 Click on Analyze and then the Hammer icon to load Autofragment and click Start. When the Autofragment Results window appears, click on the Compare Spectrum button to open the Spectrum window. If 4-chlorobiphenyl is not still present, choose File | Open File as before or click the speedbutton, and load the 4Clbiphenyl.asc spectrum file.
10 When Spectrum is called from autofragment in this manner and a spectrum file is loaded, then additional menu items are visible, and an another option appears at the bottom of the window. One new menu item is Isotope Match. If you click this item, the Isotopic Profile of the structure will be calculated and compared with the current mass spectrum. MSC will report whether an ion at the expected molecular mass is present, a calculated Match Index value, and the number of isotopes expected vs. the number found. In the case of 4-chlorobiphenyl, an ion should have been found at mass 188, with a match index of 0.9926; four significant isotopes expected, with all four found.
11 A button labeled 'Compare' is present at the bottom of the screen. The default display will have Allow H Transfers and 1H selected. Click on the Compare button. MSC Pro will compare the Autofragmentation results with the mass spectrum, and each spectral mass with a corresponding value in the Autofragmentation Results list (allowing for either 1 or 2 H transfers) will be drawn in red.
12 When Compare is finished, another menu item will appear called Summary. If you click on Summary, a report on the simple comparison results will appear. In this case, the report will indicate that there were 37 'matched' masses with an estimated match of 0.8537. This value simply provides a very rough index of overall agreement and should not be taken to represent a significant index of match likelihood
13 The option 'Print' on the Spectrum File menu will display the Spectrum Print window. You can select a printer, edit the default title, and choose whether or not to include the date and time in the printout. Click Print to print the spectrum and Close to close the window.
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