Chapter 2

2.1 Installation of the Program

For instructions on SpecMan installation, please refer to ‘The Release Notes’ or ‘specmanPCreadme’ file supplied with the program.

2.2 Spectrum Research Licensing

SpecMan and SpecManPlus are copy protected by the Spectrum Research Licensing System. This licensing system allows SpecMan/SpecManPlus to run only on the computer for which it was sold. To obtain a valid license file (license.dat) in order to activate SpecMan, please provide Spectrum Research with the Product ID from your PC system. To retrieve the Product ID, right-click on the “My Computer” icon on your Windows Desktop. Choose “Properties” from the menu that pops up, and your Product ID will be listed in the “Registered To:” section (For example: 02658-OEM-2564589-12458). Once you have received the trial license file, place a copy of it in the C:\Spectrum2001\SpecMan directory.

When the trial licensing time is nearing expiration, SpecMan will display a dialog box indicating the number of days remaining for the license. Please contact Spectrum Research for a renewal at that time.

2.3 Starting SpecMan

To launch the SpecMan program, click on the specman.exe icon from the File Manager or Windows Explorer (By default, SpecMan is installed into C:\Spectrum2001\SpecMan). The program starts with a Main Graphics Window that has a menu bar and status bar.

When SpecMan has been initiated, it attempts to locate the specman.ini file in the SpecMan directory. This file defines many of the initial settings for SpecMan, such as window sizes, default colors, font sizes, etc. (please review the ‘Edit - Initialization File’ section for more information).

2.4 The Basics

If the user is new to Microsoft Windows or Windowing systems in general, please read this section before using SpecMan to become acquainted with the SpecMan interface.

When SpecMan is first initiated, a window will appear with "SpecMan, version 2.4, (C) Spectrum Research, LLC." on the top. The area where this text appears is referred to as the ‘Title Bar.’ By pressing the left mouse button while the cursor is on the title bar, and then moving the mouse, the window will move. Releasing the mouse button will stop the window from moving. This combination (pressing the mouse button, moving the mouse, and then releasing the mouse) is known as ‘Dragging.’ Position the cursor so that it is over the word ‘File’, located immediately below the title bar. Now press and then immediately release the left mouse button. This procedure (pressing a mouse button and then releasing without moving the mouse) is known as ‘Clicking.’ The item that was clicked on was the ‘Menu Bar.’ The menu bar consists of the ‘File’ menu, the ‘Edit’ menu, the ‘Display’ menu, the ‘Analysis’ menu and the ‘Help’ menu. After clicking on the ‘File’ menu, a pulldown tab will appear consisting of additional menu items such as ‘Open Spectrum,’ ‘Open Multiple Spectra,’ ‘Open Molecule,’ ‘Save Molecule,’ etc. Menu items are the primary way that the SpecMan user can communicate with SpecMan.

In addition to the menu items on each pulldown menu, there are ‘Separator’ lines such as the line that appears above ‘Quit’ on the File menu. Its purpose is solely to make the menu easier to read. Click on the ‘Display’ menu and notice that the ‘Units’ menu item has a right pointing triangle after its text. This type of menu item is known as a pullright menu. Click the mouse on the ‘Units’ menu item and another group of menu items will appear to the right of the 'Units' menu item. This pullright feature is used to group related menu items together, reducing the size of the main pulldowns. Click on the "Display" menu and then click on the "Peaks Tables" pullright. These three menu items are known as "Toggles". Toggles have two states: "Off" (also known as "Deselected" or "Deactivated"), and "On" (also known as "Selected" or "Activated"). Click on the "1D..." toggle and another window will pop up with the title of "1D Peaks Table". Click on the "Analysis" menu and the "Peaks Tables" pullright again and now there is a check mark next to the "1D..." toggle. This means that the toggle is now "On". Click on the toggle again to turn it "Off" and the "1D Peaks Table" will disappear.

Position the mouse cursor over the frame that surrounds the entire SpecMan window. Drag the mouse to change the size of the SpecMan window. All sides of the SpecMan window can be moved to size the window. The field below the SpecMan Toolbar is known as the "Graphics Viewport". This is where information about Spectra is displayed. At the bottom of the Graphics Viewport is the "Status Bar" that lists information about what is currently happening in SpecMan. It will notify the user if the user is trying to do a task that SpecMan isn't prepared to do, and it will also give the user hints about using SpecMan.

Click on "Open Spectrum" from the "File" menu and a window will appear with the title of "Open". This type of window is known as a dialog box. While a dialog box is displayed, the user must interact with it before continuing with other areas of SpecMan. The dialog box that is currently displayed is referred to as the "File Browse Dialog" and it is used to specify a file to be opened. The "OK" button on the bottom of the dialog box is used to accept the input file. When opening a spectral data file, the user needs to input the format type for the spectral data (i.e. Varian, Bruker, JEOL, etc.). The "Files of Type:" drop down box near the bottom of the file browse dialog is used for this purpose. Varian, Bruker and JEOL sample data have been supplied with the SpecMan program, so select the file type as "Varian" and then locate the sample Varian data files in the C:\Spectrum2001\Data\Q-2 folder. The user can ascend and descend the directory structure by double clicking on a directory name from the list. (A “double click” is two clicks followed in rapid succession.) After changing to the C:\Spectrum2001\Data\Q-2\HMQC folder, click on the 'phasefile' file and then select 'OK'. The HMQC spectrum will now be displayed in the Spectral window.

To the right of the main SpecMan window is a window titled "Threshold." This is known as a "Palette." Palettes are similar to dialog boxes, however the user can interact with them and with the main SpecMan window at the same time. The "Threshold" palette is used to control the display of 2D spectral contours. Be warned, however, that changing the threshold on a large matrix can require a few minutes to compute, depending on how fast the computer is. In the "Threshold" palette, there are 4 controls known as "Sliders". The user can drag a slider bar to the left and right to raise and lower its value. Also, to the right of the "Threshold" slider is a text field that contains the currently displayed threshold value. New values can be entered into this filed and then accepted by clicking the "Update" button.

Once a spectrum is open, the user can use most of the other menu items that were previously disabled (i.e. gray and inactive).

2.5 The SpecMan Toolbar

The toolbar appears between the menubar and the Main Graphical Window, and it contains icons that represent commonly used menu items. By clicking on one of the icons, the same action that occurs when the user selects the corresponding menubar item will happen. If the user holds the cursor steady over a menubar item, a short description of that item will be displayed.

The following menu items have associated toolbar icons:

File/Open Edit/Undo

Display/Threshold Display/1D Slice

Analysis/Crosshair Cursor Display/Set Active Viewport

Display/Associate Reference Spectra Display/Set Labels

Display/Increase Zoom Display/Decrease Zoom

Analysis/Previous Zoom Analysis/Reset Zoom

Analysis/Pick Peaks Automatically/1D Analysis/Pick Peaks Automatically/2D

Analysis/Pick Peaks Automatically/3D Analysis/Show Peaks

Analysis/Move Peaks Analysis/Merge Peaks

Analysis/Add Peaks Manually/Singlet Analysis/Add Peaks Manually/Multiplet

Analysis/Add Peaks Manually/Without RefineAnalysis/Add Peaks Manually/Grid Location

Analysis/Remove Peaks Analysis/Annotate Peaks

Help/Contents

Chapter 3

SpecMan’s File Menu

This chapter helps the user navigate SpecMan’s File Menu and its features, as well as relevant main concepts and principles.

3.1 Open a Spectrum

To analyze spectra in SpecMan, first transfer the data files to the computer that SpecMan is located on, or alternately, the files can be accessed via a network. However, this latter option limits the speed of SpecMan to the speed of the network connection. The file formats that SpecMan can import are listed as follows:

· Bruker: 1rr and procs files for 1D spectra; 2rr, procs and proc2s files for 2D spectra.

· Varian: phasefile and procpar files.

· JEOL: *.als for Alice, *.nmd for Alpha, *.bin and *.hdr for Generic, *.nmdata for Lambda and *.1 for Delta.

· NMRPipe: *.ft2 files.

· NMRCompass: *.spc files.

· Galactic: *.spc files.

· JCamp: *.dx files.

· Felix: *.mat files.

· FiveAsh Database Management Handbook: *.dat files.

· Chemical Shift Database: *.sdb files.

· Chemical Concepts Database: *.jdx files.

· Custom Spectral Database: *.spd files.

· Triad: *.nmr and *.nmr_h files.

· Spectrum Research: *.exp files.

· Mass Spec (HPLC-MS) and UV (HPLC-UV): *.bin files.

· NUTS: *.xd files (x is the dimensionality).

Once the appropriate files have been transferred from a spectrometer or from a 3^rdparty processing software program, open a spectrum in SpecMan by selecting ‘Open a Spectrum’ from the File menu. Upon selecting this option, the following ‘File Browse’ dialog box is displayed:

Navigate the browser by double clicking a directory to change to it and double clicking a filename to select it and select the proper input file format from the File Type drop down menu. The user can modify the specman.ini and specmanpersonal.ini files to set the default format type to Bruker, Varian, JEOL, NMRPipe, etc., when working primarily with data of the same format.

If this is the first time that a spectrum is being read by SpecMan, the program will generate a contour file (please note that this can be time consuming on large, complex matrices, over a network, or on slow computers). After the contours have been generated, they are stored in a *.con file with the same root name as the name of the spectrum. SpecMan reads this file when the spectrum is re-loaded in the future, so that the contours don’t have to be regenerated. When transferring files that were previously analyzed by SpecMan, also copy the contour file so that the contours will not have to be regenerated.

3.2 Open Multiple Spectra

SpecMan allows simultaneous display of several spectra with multiple views. This is done by selecting, ‘Open Multiple Spectra’ from the File menu. The multiple spectra can include a combination of 1D, 2D and 3D experiments of the same sample to enable mixing and matching of any combination of 1D, 2D and 3D experiments for display. The user can also save all of the associated multiple spectral data as one file list and then open it to display the spectra. When ‘Open Multiple Spectra’ is selected, the following dialog box will appear:

Click on ‘Add’ and then select the initial spectral file from the following dialog box:

To add additional spectra, re-select ‘Add,’ and then repeat until all of the desired spectra have been added. As files are selected in the Open file browser, the list of selected files will be displayed in the Open Multiple Spectra dialog box, as shown below:

Once the desired spectra have been chosen, click 'OK' in the Open Multiple Spectra dialog box to read and display the selected multiple spectra.

At any time, only one spectrum is kept as the active spectrum and the rest are treated as passive spectra. Therefore, some of the immediate menu commands are applicable only to the active spectrum. The user can make a passive spectrum active by using the Set Active command. The user can also delete a selected file from the Open Multiple Spectra dialog box by clicking on the filename and then clicking ‘Delete.’ This delete will only delete the file from the list of multiple files and will not delete the original copy from the disk.

Multiple 2D spectra (HMQC & HMBC) are shown in the following:

When working with multiple spectra of the same molecule, it can be convenient to tie specific axes of spectra together. This is accomplished through the Tie Views option in the Display menu. When this option is chosen, the following Tie Manager dialog box is displayed:

The Tie All 1D option ties the X axes of all open 1D spectra. The Tie All 2D option ties the X axes and Y axes of all open 2D and higher spectra. The Untie All option removes all tying from all open spectra. To tie the axes of two specific spectra, select the First Spectrum and Second Spectrum text boxes to specify the desired spectra to tie. Then, click on either X&X to tie both of the X axes, or select Y&X to tie one of each axis. Note that in the tied mode the cross-hair cursors of each spectrum are also tied together so that they move together when you scroll in either spectrum. Then, select OK and the spectra will be tied as in the following (shown with the 1D reference grid lines turned off):

In the above view the HMQC spectrum is tied to the HMBC spectrum. In a similar manner, the user can also use this feature for analysis of multiple 1D spectra with tied frequency axes. This will enable the user to align common peaks between the different 1D spectra and identify multiplicities in Carbon data. Also this view mode can be used for setting the common spectral reference between Carbon and DEPT data. The user can also perform 1D peak picking and overlay the picked peaks on any related spectrum for comparison in this multiple spectral view.

3.3 Open Molecule

This function is used to display a molecule with or without its corresponding assignments. The user can import *.mdl, *.mol or *.sdf files into SpecMan and open them as shown in the following dialog box:

Open Molecule also enables the user to open multiple molecules. Click on Open Molecule to open one molecule, and then click on Open Molecule again to select a second molecule. Repeat until the desired amount of structures have been opened:

3.4 Save Molecule

This function is used to save a molecule (with assignments) as a *.mdl, *.mol or *.sdf file. Select ‘Save a Molecule’ from the File menu and the following dialog box will appear:

Select the desired format and type in a filename to save the structure file. Note that the assignments will be appended to the end of the structure file.

3.5 Manage Custom Database

This function is used for creation of a custom spectral database. The user can create a database from any type of vendor data that can be imported by SpecMan, and can then search through individual spectra, structures, peaks tables and assignment tables. More details regarding the features of assignment tables generated by NMR-SAMS can be found in the Display Menu of this document.

To create a database, first select Manage Custom Database from the Edit menu, and the following window will appear:

To create the database in the current directory, type a name in the text box next to Database Filename. However, to create the database in a different directory, select the Browse button next to the Database Filename text box. After selecting the new directory, type in the name of the database and the *.spd filename will be added automatically.

After defining the database name, the next step is to add the entries. Each entry can consist of spectra, molecular structures, peaks tables, and assignment tables. Except for the spectrum, the other entries are optional. If for instance a peaks table or assignment table is not designated, then the peaks table or assignment table window will not be displayed. When 'Add' is selected, the following open file selector will appear to aid the user in finding a 1D or 2D spectra:

Once the correct spectrum file has been selected, the path name corresponding to the chosen spectrum will be highlighted in the Spectra text box on the Manage Custom Database window. Select browse next to the Molecule text box in order to select the molecule (*.mdl , *.sdf and *.mol files from NMR-SAMS, along with other *.mol structures created by sketching programs such as ChemDraw, ISIS, etc.). Then type in the name of the molecule, and select browse next to the Peaks Table text box. This will allow the user to select the desired peaks table (with extension *.pks). Then, select browse next to the Assignment Table text box and select the corresponding assignment table (with extension *.rst). The completed initial entry will appear as follows:

To enter additional entries, click Add and repeat the above-mentioned process. All of the information is stored in memory until 'Create' is clicked. When 'Create' is clicked, the information is written to an *.spd file. If the user selects Cancel before Create, all entered information will be lost. The user's custom database has now been created, and can be accessed by selecting Open a Spectrum under the File menu. Be sure to select the type of file as *.spd, and the following windows will appear:

The Database Browser is used to browse through and search for entries in the database. The browser contains the name of the current spectrum; a slide bar for sliding through the individual entries in the database; the number of entries in the database, and the current entry being displayed; a text block where the user can type in the name (or a portion of the name) of an entry to search for; two radio buttons which enable searching either for the name of a spectrum or the name of a molecule; and a listing of all of the entries in the database. It is important to note that the molecule name can include whatever information regarding the sample that the user desires. For example, the molecule name can include the experiment number, or the date on which the data was collected and analyzed, etc. The spectrum name can also be modified by selecting ‘Set Labels from the Edit Menu. The following dialog box will be displayed:

If after creating the database the need to edit the database arises, the entries can be edited by selecting Manage Custom Database from the File menu, or by selecting Edit Custom Database from under the Edit menu (More details about this can be found in the Edit -Custom Database section of this document). SpecMan allows the user to open multiple spectra with one spectrum as an *.spd file and another as a non-*.spd file. One application of this feature is to manually search and compare the spectrum of an unknown with the known entries in the database.

3.6 Import NMR-SCAPE Database

This option is used for importing and displaying the *.mba files that are generated by Spectrum Research’s ¹³C spectral prediction program, NMR-SCAPE. By selecting ‘Import NMR-SCAPE Database,’ the following dialog box will appear:

Select the NMR-SCAPE *.mba file and then click ‘Open’ and the following dialog box will appear:

In order to be able to view the *.mba file in SpecMan, the file needs to be converted to a *.jdx file format, so enter a File Name and click ‘Save.’ To view the *.jdx file, select ‘Open Spectrum’ from the File Menu.

3.7 Save Multiple Spectra

This option is used for saving the current list of displayed multiple spectra. This enables the user to save a selected list of spectral data that can be accessed easily during a future session for analysis. Upon selecting this option, the following dialog box will appear:

Enter the output file name, and the list will be save with the extension *.list. The next time that the user opens multiple spectra, this file can be opened and all of the spectra listed in this file will be displayed.

3.8 Prepare NMR-SAMS Data

SpecMan can now prepare data for input into NMR-SAMS. The following options are available under the pull-right menu of Prepare NMR-SAMS Data:

By selecting Define Molecular Formula, the following dialog box will appear:

Select 'Browse' to define the location for saving the NMR data and then type a filename (the program will automatically add the *.nmr extension). Finally, input the molecular formula of the sample, or leave it as “Unknown” if the exact formula is not known.

The next step in preparing the NMR-SAMS data, is to convert the H-1 Peaks List into the proper format for incorporation into NMR-SAMS. The following dialog box will appear when 'H-1' is selected from the pull-right menu:

Select the location of the H-1 peaks table, and the peak list will be converted and written into the *.nmr file. A *.mdf file is also created which contains the empirical formula information. In a similar manner, the peak lists for C-13 and DEPT; COSY; HMQC (or HETCOR); HMBC (or COLOC); NOESY (or ROESY); and INADEQUATE can be converted (for more details, refer to the Q2 or Taxol Tutorials). This function originally resided only in NMR-SAMS, but is now also found in SpecMan. If a problem or error arises during data conversion, it is now possible to compare the converted data with the original spectrum and peak list information right in the SpecMan program. If the need to edit the converted data arises, select NMR-SAMS data from the Edit Menu. More details regarding the editing of NMR-SAMS data can be found in the Edit Menu portion of this document.

3.9 Export

SpecMan can export spectra as Postscript (*.psf), GIF (*.gif), or Acrobat (*.pdf) files. Select ‘Export’ and the following dialog box will appear:

It is important to note that only the spectra in the active spectrum window will be exported. You will need to make other spectral windows active before you can export their contents.

3.10 Print

SpecMan now has the ability to print spectra, structures, peak tables and assignment tables. The following options are available from the pull-right menu of Print:

The active spectrum, the spectral windows (1D, 2D), the active molecule, the molecule window, the peaks tables (1D, 2D, 3D), and the assignment table can all be sent directly to the printer.

3.11 Save Submatrix

This option is used for saving the current 2D spectral view as a new spectral data set. This enables the user to save selected spectral regions. Prior to selecting this option a zoom may be performed. When 'Save Submatrix' is selected, the following ‘Save As’ dialog box appears:

Type a filename to define the folder name in which the submatrix will be saved with appropriate parameter files. Click 'Save' to save the current expanded view as new spectral data. To save the transposed view of the spectrum, select ‘Transpose Spectrum’ from the File Type option and then click 'Save'.

3.12 Save Projection

This option is used for saving the 1D X or Y projection of 2D spectra. When this option is selected, the following ‘Save As’ dialog box appears:

This allows the user to save either the X or Y projection. Once a filename has been defined and the 'Save' button has been clicked, a folder with the filename will be created and the projection will be saved in this folder as a new spectral data file. In addition, a *.prj file will be created in the original 2D spectral data folder.

3.13 Quit

To exit SpecMan, use the File/Quit menu option. If there are peaks tables open, SpecMan will ask if the user wants to save them before exiting.

Chapter 4

SpecMan’s Edit Menu

This chapter helps the user navigate SpecMan’s Edit Menu and its features, as well as relevant main concepts and principles. The Edit menu lists options related to the editing of spectral data into and out of SpecMan. The Edit menu also contains enhanced clipboard functions for report generation.

4.1 Undo

This option allows the user to reverse a previous action. After performing an action that the user wants to reverse, select ‘Undo’ from the ‘Edit’ menu, and the action will be undone. Select the 'Redo' option to re-perform the action that was just reversed and things will return back to the previous display/action. The following are a sampling of the actions that can be undone:

· Manual Peak Addition (All Options)

· Merge Peaks

· Move Peaks

· Remove Peaks

Hint: The text of the ‘Undo’ option changes to reflect the current action that can be undone.

Warning: Only the most recent action will be undone, so if an action is perform after the error, SpecMan will not be able to undo the error.

4.2 Copy Spectrum

Select Copy Spectrum from the Edit menu to copy the spectrum in the SpecMan window and the following pull-right menu will appear:

The user can choose to copy just the active spectrum (if multiple spectra are open), or the entire contents of the 1D spectral window or the 2D spectral window by selecting from the three pull-right options.

Once the spectrum/spectral windows have been copied, open up a Word document side by side with SpecMan. Select Paste from the Edit menu in the Word document to paste the spectrum into the Word document as shown below:

4.3 Copy Molecule

Choose Copy Molecule from the Edit menu to copy the molecule in the SpecMan window. The following pull-right options are available:

The user can choose to copy the active molecule (if multiple molecules are open), or the entire contents of the Molecule Window by selecting from the two pull-right options.

Once the molecule/molecule window has been copied, select Paste from the Edit menu in Word to paste the molecule into the Word document.

4.4 Copy Assignment

Select Copy Assignment from the Edit menu to copy the assignment table in the SpecMan window. Then, open a Word document side by side with SpecMan. Select Paste from the Edit menu in Word to paste the assignment table into the Word document. The assignment table will appear in text format in the Word document. In order to convert the assignment table into actual table format, highlight the text portion of the assignment table, and under Word’s Table menu, select Convert Text to Table and the following will appear:

4.5 Copy Peaks

Select Copy Peaks from the Edit menu to copy the peaks table in the SpecMan window. The following pull-right options are available:

The user can choose to copy the 1D, 2D or 3D Peaks Tables. Once a Peaks Table has been copied, select Paste from the Edit menu in Word to paste the table into the Word document.

The peaks table will appear in text format in the Word document. In order to convert the peaks table into actual table format, highlight the text portion of the assignment table, and under Word’s Table menu, select Convert Text to Table and the following will appear:

4.6 Initialization File

SpecMan allows the user to customize many of the display attributes to meet each individual's display and interaction needs. Among these are window sizes, fonts and colors, default values for parameters in the threshold palette, and other attributes related to cursors and tables. This information is stored in the specman.ini and specmanpersonal.ini files which should always remain in the SpecMan directory. These files are plain English text files and can be edited with any text editor. SpecMan reads the specman.ini file when the user first initiates the program.

SpecMan allows the user to customize its display by editing the specmanpersonal.ini file. To do so, select ‘Initialization File - Edit Personal Ini’ from the Edit Menu. This will bring up the following:

The details of the specmanpersonal.ini file format are: Lines that begin with a pound sign (#) are comments which are ignored by the program. The other lines are of the format: <keyword> <value(s)>, where <keyword> tells SpecMan what default is being set, and <value(s)> is one or more numbers or letters that the default is being set to. The actual number and type of values are determined by the individual defaults. For example, the MainWindow keyword sets the location of the Main SpecMan window on startup. It requires 2 integers specifying the x and y locations of the window. Therefore, its line looks like: 'MainWindow 20 50', which puts the Main Window's upper left corner at the screen coordinate (20, 50).

The user can also choose to toggle between the black default ini and the white default ini files. These are files supplied with SpecMan that contain different color selections for backgrounds (a white background that is useful when copying and pasting spectra, molecules, etc., and a black background).

4.7 Database File

This feature allows a custom database file (an ASCII file) to be displayed in Notepad as shown below:

The % Version is for our internal usage, so do not alter this line or the line below it, while the % Num lists the number of total entries in the custom database. Lines beginning with % cannot be edited, while the rest of the lines can be modified.

4.8 NMR-SAMS Data

This feature allows a NMR-SAMS Data file (*.nmr file) to be displayed in a text editor for editing, as shown below:

4.9 Set Labels

SpecMan allows the user to change the spectral labels and title. To do so, select ‘Set Labels’ from the Edit menu and the following dialog box will appear:

Enter the desired new labels and then click ‘OK’ to initiate the change. Note that this will only affect the active spectrum (if multiple spectra are open). To change the labels of other spectra, first set each spectrum as active and then select Set Labels.

4.10 Spectral Parameters

When SpecMan imports processed spectrum files, it reads the data's spectral parameters, such as spectrometer frequency, data size, sweep width, etc. Although these parameters are obtained from the original data, the user can modify them by selecting ‘Spectral Parameters’ from the Edit menu.' The following dialog box will appear with the current spectral parameters listed (for example, for a 1D spectrum):

Depending on the dimensionality of the experiment, the dialog box will show parameters that correspond to different spectral dimensions. For example, the parameters for 1D spectra will have only the X dimension values, while the parameters for 2D spectra will have values for the X and Y dimensions. The following spectral parameters dialog box will be displayed for a 2D spectrum:

Dialog boxes associated with other types of spectral data (MassSpec and UV, respectively) are also shown below:

After changing the spectral parameters, click 'OK', and the new parameters will be saved in the data file for future use.

4.11 Set Reference

A spectral reference is a peak corresponding to a known reference from which other frequencies are measured. When ‘Set Reference’ is selected from the Edit menu, SpecMan displays a crosshair cursor and prompts the user to click on a peak that can be used for setting the reference. Upon clicking on a 1D reference peak, the following dialog box appears:

Next, enter the new reference points and reference values. Depending on the dimensionality of the data, the user will need to enter various reference points and reference values. For example, 1D spectra only require the X coordinate, while 2D spectra require X and Y point coordinates and values, and 3D spectra require X, Y and Z point coordinates and values, as shown below:

2D:

3D:

Dialog boxes associated with other types of spectral data (MassSpec and UV, respectively) are also shown below:

Click 'OK' to apply the new reference, and SpecMan will store the new reference values in the spectral data file and update the display with the new scales corresponding to the new reference frequency.

How to Set Reference on a 1D peak with respect to another reference peak in a related 1D spectrum:

This option enables the user to set reference on a peak graphically without having to enter reference values. For example, this method can be used for setting reference on a DEPT spectrum with respect to peaks picked in a ¹³C spectrum. To do this, zoom in on a peak in the DEPT spectrum to set reference on, and then load a ¹³C peaks table with which you wish to match the reference. Next, select ‘Set Reference’ from the Edit menu, and place the cursor on the DEPT peak to align with the peak label of the ¹³C peak. Click the left mouse button and keeping it pressed (a symbol consisting of a circle surrounding a ‘+’ sign will appear), drag the symbol until the symbol lines up with the ¹³C peak label. Release the button and the set reference dialog box will appear.

Internally, SpecMan computes the reference values with respect to the peak label, and updates the corresponding X values in the dialog box. Click ‘OK’ to accept the new reference, and the spectrum scales will be updated with the new values. In this manner the user can align the 1D DEPT with the 1D ¹³C spectrum to maintain same spectral reference.

How to Set Reference on a 2D peak with respect to a grid line intersection based on 1D reference peaks:

This option enables the user to set reference on a 2D peak graphically without having to enter reference values. To do this, the user needs to zoom in on the peak and the grid line intersection to match. Next, select ‘Set Reference’ from the Edit menu, and place the cursor on the center of the 2D peak to align. Click the left mouse button and keeping it pressed, drag the symbol (a circle with a ‘+’ inside it) until the symbol lines up with the grid line intersection. Release the button and the set reference dialog box will appear.

Internally, SpecMan computes the reference values with respect to the grid line, and updates the corresponding X and Y values in the dialog box. Click’ OK’ to accept the new reference, and the spectrum scales will be updated with the new values. In this manner the user can align 1D reference spectrum with a 2D spectra.

4.12 Molecule

The user can edit an active molecule by selecting ‘Molecule’ from the Edit menu. This will bring up the following Molecular Editor palette:

After the Molecular Editor is displayed, the user may need to click ‘Clear’ to remove the current displayed structure, or the user can build a structure starting with the current displayed structure. To sketch the target structure, first select Add, Atom, and Continuous Mode. Leave Element as “C”, and Ambiguous unchecked (this toggle is reserved for defining a substructure, and is currently not used). Then click in the main graphics window at the position where the first atom is supposed to appear. An atom will be drawn at that location and when the next location is clicked, another atom will be drawn with a bond connected to the previous atom. In this manner atoms are added at the locations where the user clicks and bonds are added automatically between the current and last atom. To discontinue the addition of an automatic bond between atoms, turn off the Continuous Mode.

Once the skeleton has been sketched, the user can modify the structure. To modify an atom, select Modify and Atom, and then type the desired element symbol after Element. The user can also move the slider to change the valence of the atom (i.e., choose a valence different than the default). Then click on the atom to change. To modify a bond, select Modify and Bond, and then select the desired bond type. If the user is not sure about the connectivity or attached protons of an atom, select Ambiguous. Next, click on the two associated atoms of the bond to modify, and the bond will be modified. If Continuous Mode is on, use the middle (or right) mouse button to pause the continuous mode temporarily.

To delete an atom, select Delete and Atom, and then click on the atom to delete. To delete a bond, select Delete and Bond, and then click on the two associated atoms of the bond to delete. After building the target structure, click OK to accept it.

To renumber an atom, select Renumber from the Molecular Editor dialog box. This feature will enable the user to renumber atoms in a structure to conform to IUPAC or other conventions. Open a molecule along with its corresponding assignment table, as shown below:

Next, select Renumber in the Molecular Editor dialog box, and the dialog box will appear as shown below:

Select the number to begin renumbering with (for example, 1), and type it in the text box next to Renumber. Then, click on the first atom in the molecule (for example, atom 26) to renumber as atom 1. Internally, atom 26 will now become atom 1, and the old atom 1 will be renumbered as atom 26. Additionally, the assignments will also be swapped so that they remain with the correct atom, and the assignment table will be updated with the new atom numbers. When another atom is clicked on, the program will increment the initial renumbering number. For example, the next atoms will be labeled two, three and so on. If the user had begun renumbering with 20, then the next atoms would be labeled 21, 22, 23, etc. The renumbering of atom 26 is shown in the following window:

Comparison of the initial molecule (shown on Page 41) with the above modified molecule shows the old atom number 26 now renumbered as atom 1.

Chapter 5

SpecMan’s Display Menu

The Display menu lists options related to the graphical display of SpecMan. This chapter helps the user navigate SpecMan’s Display Menu and its features, as well as relevant main concepts and principles.

5.1 Set Active Viewport

When multiple spectra are open, SpecMan allows the user to set one spectrum as active (referred to as an active viewport) and all functions will be applied to the active spectrum. To set a spectrum active, select ‘Set Active Viewport’ form the Display menu and click on the desired spectrum, and it will become active. The color of the axes will be changed to reflect that the spectrum is now active (Please note that the color of the axes is controlled by the specmanpersonal.ini file’s ‘FrameColor’ and ‘InactiveColor’ options).

Please note that there are several menu items that do not require user input, such as the zoom options, that can be applied to any open spectrum. To use such functions, simply click in the desired viewport.

5.2 Swap Viewports

By default, the order of displayed viewports is the order that the 2D spectra were opened in. To change this without reopening the spectra, select ‘Swap Viewports’ from the Display menu, and then click on the two viewports that you wish to swap. The two viewports will then be redrawn with the reordering of the 2 viewports. In this manner, any viewport order can be achieved through subsequent selection of ‘Swap Viewports.’

5.3 Threshold

The Threshold palette is used for controlling the noise threshold used in the generation of contours for the spectral display. The ‘Threshold’ option from the Display menu acts as a toggle that displays and hides the threshold palette, as shown below:

The controls in this palette can be used to control the following:

· Adjust the noise threshold for contouring by specifying a value or by dragging the slider bar.

· Adjust the contour level separation. This specifies the factor by which the base threshold is multiplied to determine the cutoff of each contour level.

· Adjust the maximum number of contour levels to be drawn. The maximum levels allowed are 100. (If the cutoff of a level exceeds the maximum intensity of the data points, then the maximum number of levels is automatically set to the level that corresponds to the previous cutoff).

· Define the starting level for display. This feature allows stepping through the available contour levels. Performing this activity can assist in resolving partially overlapped peaks and also locating peak tops.

· Define the type of contour levels (whether positive, negative or both) to be drawn.

The Reset button returns the palette values to the defaults.

If Auto Redraw is checked, then any change to the palette will be reflected immediately. Otherwise, contouring will only take place when the update button is checked. All operations described above will only be applicable to the active spectrum. If the user wishes to change the display attributes of another spectrum in multiple view mode, first set the interested spectrum as active. In the case of a 3D spectrum, select the ‘Apply to all Tiers’ option in the Tier Palette to regenerate contours for all tiers with the same threshold. When multiple spectra are displayed, the Threshold palette is associated only with the active spectrum.

5.4 2D Viewport Layout

By default, when displaying multiple 2D spectra, viewports are displayed in a row-based square. The following options are available from the ‘2D Viewport Layout’ pullright menu:

Row-based square

One row

Column-based square

One column

Depending on the type of spectra that the user is working with, the user can change this to be one row, one column, or a column-based square. To do this, select the desired layout from the ‘Viewport Layout’ menu option on the ‘Display’ menu. The layout will be immediately changed to the selected layout. The user can also change the default selection by modifying the ‘ViewportLayout’ entry in the specmanpersonal.ini file. Note that 1D viewports are always displayed in one column, regardless of the setting of the viewport layout.

5.5 Units

This feature allows the user to select the units for the X and Y axes. By selecting ‘Units’ from the Display menu, the following putllright menu will appear:

Select PPM, Data Points or Hertz to display the desired units.

5.6 Tie Views

When working with multiple spectra of the same molecule, it can be convenient to tie the axes display. This can be accomplished by selecting ‘Tie Views’ from the ‘Display’ menu. When this option is chosen, the following Tie Manager dialog box will be displayed:

The Tie All 1D option ties the X axes of all open 1D spectra. The Tie All 2D option ties the X axes and Y axes of all open 2D and higher spectra. The Untie All option removes all tying from all open spectra. To tie the axes of two specific spectra, select the First Spectrum and Second Spectrum text boxes to specify the desired spectra to tie. Then, click on either X&X to tie both of the X axes, or select Y&X to tie one of each axis. Note that in the tied mode the cross-hair cursors of each spectrum are also tied together so that they move together when the user is scrolling in either spectrum. Then, select OK and the spectra will be tied as in the following:

In the above view, the ¹³C spectrum is tied to the DEPT-135 spectrum. This enables the user to align common peaks between the different 1D spectra and identify the multiplicities in the Carbon data. Also this can be used for setting the common spectral reference between Carbon and DEPT data. The user can also perform 1D peak picking and overlay the picked peaks on any related spectrum for comparison in this multiple spectral view.

In a similar manner, one can also use this feature for analysis of multiple 2D spectra with tied frequency axes.

When working with multiple 2D spectra the following Tie Manager dialog box will appear:

Click ‘OK’ and any 2D experiments (such as HMBC and HMQC) will have tied axes as shown below:

When working with multiple 3D or 4D spectra (as in SpecManPlus), the following Tie Manager dialog box will appear:

5.7 Overlay

When comparing two similar peaks, it can be beneficial to the user to have the option of overlaying the two spectra for closer comparison. When selecting ‘Overlay’ from the Display menu, the user can choose to overlay with an offset (adds an overlay ppm value to the overlaid spectrum which can be useful when the ppm regions of the peaks don’t match exactly), or without an offset (overlays according to the spectral ppm regions, with no offset added).

To overlay with a ppm offset, select ‘Overlay - Overlay with Offset’ from the Display menu and then click on a spectrum to overlay on the Active Spectrum. A dialog box will appear that prompts the user to enter ppm offsets for the X and Y dimensions. Entering zero (0) for a dimension results in no overlay for that dimension. Click ‘OK’ and the overlaid spectrum’s viewport will disappear and the spectrum will be drawn in the active Viewport (with the ppm values of the overlaid spectrum shifted by the specified offset). The zoom features will now zoom both spectra at the same time. Reselection of ‘Overlay - Overlay with Offset’ will remove the overlay.

To overlay without an offset, select ‘Overlay - Overlay without Offset’ and then click on a spectrum to overlay on the Active Spectrum. The overlaid spectrum’s viewport will disappear, and the spectra will be drawn in the active Viewport. The following shows the original HMBC and HMQC spectra and then the overlay of the HMQC spectrum (red) on the HMBC spectrum (black):

Reselection of ‘Overlay - Overlay without Offset’ will remove the overlay.

Note: The overlaid spectrum is displayed in a different color (red). The default spectral colors are controlled by the ‘ExpPosColor’ and ‘ExpNegColor’ initialization file options. The spectral colors of overlaid spectra are controlled by the ‘SecondPosColor’ and ‘SecondNegColor’ initialization file options.

5.8 Associate Reference Spectra

SpecMan enables the user to display 1D reference spectra and their peak lists on a 2D spectrum for quick identification of missing and overlapping peaks, and it is also used in grid-intelligence based peak picking. By selecting ‘ Associate 1D Reference Spectra’ from the Display menu, the following will appear:

To associate a reference spectrum (displayed along the axes of the 2D spectrum), check the box next to ‘1D Reference Spectral Along X Axis’ and next to ‘1D Reference Spectral Along Y Axis’ and then select the appropriate 1D spectral files. To display each spectrum’s peak list (displayed as grid lines on the 2D spectrum), check the box next to ‘1D Reference peak List along X Axis’ and ‘1D Reference peak List along Y Axis’ and then select the appropriate 1D peak files.

Click ‘OK’ and the following will be displayed:

The association of reference spectra also aids in the peak picking process by identifying the location of potential 2D peaks at the intersection of the 1D peak lists’ gridlines (drawn parallel to X and Y axis). SpecMan uses a novel gridline-based auto peak picking algorithm to efficiently pick peaks in 2D spectral data. This algorithm is particularly useful for peak picking the center of mass of crosspeak multiplets (for example, in 2D COSY, HMBC, etc.). The details of using gridline-based peak picking are located in the ‘Analysis – Pick Peaks Automatically – 2D’ section of this user guide.

The user also has to ability to associate a Projection, Row, and/or Column of the current 2D spectrum. To associate a Projection, check the box next to ‘2D Projection along X axis’ or next to ‘2D Projection along Y axis.’ Click ‘OK’ and the projection of the 2D spectrum will be displayed. To view a particular row or column, check the box next to ‘Row’ or ‘Column’ and enter the desired row/column numbers in the text box.

Please note that information regarding associated 1D reference spectra and peak lists, along with other attributes are saved in an environment file (*.env) in the same directory in which the original spectrum is located. Therefore, the next time the spectrum is opened, the environment file will be read and the 2D spectrum will be displayed along with the 1D reference spectra, lines, projections, etc.

Adjusting the vertical scales of reference 1D spectra

Once the reference spectra have been displayed along the X and Y axes, the vertical scaling of the 1D reference peaks can be adjusted by moving the arrow or crosshair cursor to an intensity level (with respect to some strong 1D peak) on the 1D spectrum along the X axis (or Y axis), and click at the desired level. All of the 1D peaks will be enlarged vertically since the vertical scales have been changed. To reset the scaling to the original display, move the cursor to a point below the base line of the 1D spectra and click the left mouse button. In a similar way, the 1D reference along the Y axis can also be re-scaled.

SpecMan also allows the user to modify the maximum intensity of the Associated 1D Reference Spectra (useful if the 1D spectrum is of low concentration and the solvent peaks are much more intense than the peaks of interest).

To set the intensity cutoff, click on a point that will then become the new maximum, and the 1D spectrum will be redrawn. The user may reset the cutoff back to the highest point by clicking above the 1D drawing area, or between the 1D drawing area and the 2D axes boundary.

5.9 1D Slice

All 1D spectra are displayed in a window entitled, ‘1D Slices.’ The user can display or hide the 1D slice window by selecting and de-selecting ‘1D Slice’ from the Display menu.

When the 1D slice window is displayed, a ‘1D Control Panel’ will also appear, as shown below:

Prior to auto or manual 1D peak picking, the appropriate threshold level can be set by selecting ‘Set Threshold’ from the 1D Control Panel. Next, place the cursor in the 1D slice window, and a red, horizontal line will appear. Move the red, horizontal line to an appropriate level for the cutoff threshold for 1D peak picking and click the left mouse button. The corresponding threshold value will be updated in the 1D Control Panel, and this value will be used for peak picking. When the 1D automatic peak picking option is selected, this threshold value will appear in the peak picking dialog box as the default threshold.

The 1D slice window is also used to display 1D traces from a 2D or 3D spectrum. When a 2D spectrum is displayed in the main window and ‘Display – 1D Slice’ is selected, the user can click on a cross peak with the crosshair cursor, and the 1D traces of rows and columns corresponding to this location will be displayed in the 1D slice window, as shown below:

Similarly, when working with a 3D spectrum, all three 1D traces (rows, columns and tiers) can be displayed by clicking the crosshair cursor on a cross peak in a 2D tier plane of a 3D data.

When the 1D slice window is displaying 1D traces from a 2D or 3D spectrum, the following ‘1D Control Panel’ will appear:

5.10 Molecule

This feature allows the user to display different features of a molecule. When Molecule is selected under the Display menu, the following pull-right options are available:

Window - This will allow the user to close or display the molecule window.

Chemical Shifts - This will allow the user to display chemical shifts related to the molecule.

Carbon Symbols - This will allow the user to display the atoms with their Carbon symbol.

Atom Numbers - This will allow the user to display the atom numbers.

Balls - This will allow the user to view the molecule with the atoms as balls.

Protons - This will allow for the protons to be visible.

Molecular Formula - This will allow the molecular formula to be visible.

Molecular Weight - This will allow the molecular weight to be visible.

Refine - This option allows the user to refine the display of the molecule.

5.11 Peaks Table

This option allows the user to display or hide the 1D and 2D Peaks Tables that are associated with spectral data by selecting and reselecting ‘Peaks Table – 1D/2D’ from the Display menu. A 1D Peaks Table is displayed below:

The details for each row in the 1D peaks table are listed below:

Column Name	Description
ID	This represents a unique number that SpecMan uses for book keeping
Label	This represents the label assigned to a peak by the user. This label is used for annotating the peak in the spectral view.
X Point	This represents the Data point location of the peak along the X axis. It corresponds to the actual data point in the spectrum.
X Value	This represents the frequency/chemical shift value in the appropriate units for the X data point in the spectrum. These numbers are based on the spectral reference that has been used.
Multiplicity	Multiplicity of the peak. Currently, all peaks have the Multiplicity of u (unknown). This column is reserved for future expansion.
Intensity	This represents the intensity of the peak. These intensities can also be used for NOE distance calibration of NMR spectra.
File	This represents the Spectral Data File that generated the list of 1D picked peaks. For peaks that have been picked on 1D rows or columns the word Row or Column is printed in this column

A 2D Peaks Table is displayed below:

Each entry in the table represents a single peak. The details of the columns in each row of the 2D table are given below:

Column Name	Description
ID	This represents a unique number that SpecMan uses for book keeping
Label	This represents the label assigned to a peak by the user. This label is used for annotating the peak in the spectral view.
X Point	This represents the Data point location of the peak along the X axis. It corresponds to the actual data point in the spectrum.
Y Point	This represents the Data point location of the peak along the Y axis . It corresponds to the actual data point in the spectrum.
X Value	This represents the frequency/chemical shift value in the appropriate units for the X data point in the spectrum. These numbers are based on the spectral reference that has been used.
Y Value	This represents the frequency/chemical shift value in the appropriate units for the Y data point in the spectrum. These numbers are based on the spectral reference that has been used.
J Coupling	J coupling constant for this peak. Currently, all peaks have the J Coupling value of 0. This column is reserved for future expansion.
Multiplicity	Multiplicity of the peak. Currently, all peaks have the Multiplicity of u (unknown). This column is reserved for future expansion.
Intensity	This represents the intensity of the peak. These intensities can also be used for NOE distance calibration of NMR spectra.
File	This represents the Spectral Data File which generated the list of 2D picked peaks. For peaks that have been picked on 1D rows or columns the word Row or Column is printed in this column.

A 3D Peaks Table (available in SpecMan+) is displayed below:

Each entry in the table represents a single peak. The details of the columns in each row of the 3D table are given below:

Column Name	Description
ID	This represents a unique number that SpecMan uses for book keeping
Label	This represents the label assigned to a peak by the user. This label is used for annotating the peak in the spectral view.
X Point	This represents the Data point location of the peak along the X axis. It corresponds to the actual data point in the spectrum.
Y Point	This represents the Data point location of the peak along the Y axis. It corresponds to the actual data point in the spectrum.
Z Point	This represents the Data point location of the peak along the Z axis. It corresponds to the actual data point in the spectrum.
X Value	This represents the frequency/chemical shift value in the appropriate units for the X data point in the spectrum. These numbers are based on the spectral reference that has been used.
Y Value	This represents the frequency/chemical shift value in the appropriate units for the Y data point in the spectrum. These numbers are based on the spectral reference that has been used.
Z Value	This represents the frequency/chemical shift value in the appropriate units for the Z data point in the spectrum. These numbers are based on the spectral reference that has been used.
J Coupling	J coupling constant for this peak. Currently, all peaks have the J Coupling value of 0. This column is reserved for future expansion.
Multiplicity	Multiplicity of the peak. Currently, all peaks have the Multiplicity of u (unknown). This column is reserved for future expansion.
Intensity	This represents the intensity of the peak. These intensities can also be used for NOE distance calibration of NMR spectra.
File	This represents the Spectral Data File that generated the list of 3D picked peaks. For peaks that have been picked on 3D bricks from a 4D spectra, the brick PPM is printed in this column.

Edit Peaks Table

SpecMan allows the user to edit the peaks tables by selecting ‘Edit Table’ from the 1D Peaks Table. The following ‘Edit Peaks Table’ dialog box will appear:

This dialog box allows the user to sort the peak entries in ascending/descending order according to ID, X Point, X Value or Intensity, and to renumber the peak entries in the peak tables.

By selecting ‘Edit Table’ from the 2D Peaks Table, the following ‘Edit Peaks Table’ dialog box will appear:

This dialog box allows the user to sort the peak entries in ascending/descending order according to ID, X Point, Y Point, X Value, Y Value, or Intensity, and to renumber the peak entries in the peak tables. In addition, the user can also extract the X or Y coordinates to create a 1D peaks table.

5.12 Assignment Table

This feature allows the user to display or hide the assignment table corresponding to the active molecule. The assignment table consists of spectral data arranged in columns. The first column lists the labeled number of heavy atoms in the structure, while the second column shows the atomic symbols of heavy atoms (with attached protons). The third column lists the ¹³C shifts, while the fourth column depicts the ¹H shifts.

In order to view the assignment table, open a spectrum and a molecule, and make sure to select the molecule window as Active. Then, select ‘Assignment Table’ from the Display menu, and the following will appear:

It is also sometimes desirable to display a blank assignment table, for example, when the user is manually assigning chemical shifts. For more about manual assignment, review the section on Manual Assignment under the Analysis Menu. Remember to select Save after any additions to the blank Assignment Table, or they will be lost if you quit SpecMan or close the assignment table.

5.12 Increase Zoom

SpecMan allows the user to increase the zoom while retaining the X/Y sweep width ratio by selecting ‘Increase Zoom’ from the Display menu. This will enable the user to zoom in by a factor of 2, thereby eliminating the need for the use of the default rubberband zoom.

Please note that to keep the vertical expansion stationary and change only the horizontal scales while using the default rubberband zoom, the user can keep the ‘Shift’ key pressed while doing the rubberband zoom. The horizontal limits will change, while the vertical scales of the zoom box remain unchanged. To keep the horizontal scales stationary and zoom along the vertical scales, keep the ‘CTRL’ key pressed while applying the rubberband zoom. This feature is useful in expanding the vertical scales of weak 1D peaks.

5.13 Decrease Zoom

SpecMan allows the user to decrease the zoom while retaining the X/Y sweep width ratio by selecting ‘Decrease Zoom’ from the Display menu. This will enable the user to zoom out by a factor of 2, thereby eliminating the need for the use of the default rubberband zoom.

5.14 Mirror Zoom

This option enables the user to swap the X and Y ppm zoom limits of homonuclear spectra. Select ‘Mirror Zoom’ from the Display menu and the X and Y zoom limits will be interchanged.

5.15 Zoom Manually

This option allows the user to expand the spectral view to a specific size. When ‘Zoom Manually’ is selected from the Display menu, the following ‘Manual Zoom’ dialog box will appear:

The dialog box contains the current zoom limits, and prompts the user to enter new zoom limits for the current 1D spectral view. Once done, click ‘OK’ to accept the zoom limits, and the spectral view will be updated to the new expansion. Note: The user can zoom out beyond the full spectral extents, however the X/Y minima must always be smaller than the maxima.

When a 2D spectrum is displayed, the following ‘Manual Zoom’ dialog box will appear:

When analyzing other spectral data, the following analogous dialog boxes will be displayed. Mass Spec and UV examples, respectively, are shown below:

5.16 Previous Zoom

This option allows the user to return to the previous zoom limits. It acts as a toggle and can be used to toggle between the last two zoom limits that were applied. When a 1D spectrum or multiple 1D spectra are displayed, the user needs to click in the 1D slice window after selecting ‘Previous Zoom’ from the Display menu to reset the zoom limits. When a 2D spectrum is open, it is not necessary to click on the 2D spectrum, unless multiple 2D spectra are open.

5.17 Reset Zoom

SpecMan allows the user to reset the expansion to the original zoom limits by selecting ‘Reset Zoom’ from the Display menu. When a 1D spectrum or multiple 1D spectra are displayed, the user needs to click in the 1D slice window after selecting ‘Previous Zoom’ from the Display menu to reset the zoom limits. When a 2D spectrum is open, it is not necessary to click on the 2D spectrum, unless multiple 2D spectra are open.

Chapter 6

SpecMan’s Analysis Menu

The Analysis menu lists the options related to spectral analysis. This chapter helps the user navigate SpecMan’s Analysis Menu and its features, as well as relevant main concepts and principles.

6.1 Peak Picking Options

SpecMan allows the user to select one of three different methods for determining the placement of a peak multiplet location. Select ‘Peak Picking Options’ from the Analysis menu and the following pullright menu will appear:

Select ‘Average’ to perform a straight average of the peak locations. Select ‘Weighted Average’ to weigh the peak locations with their intensities. Select ‘ Highest Peak’ to locate the peak on the intensity of the largest magnitude within the multiplet region.

6.2 Pick Peaks Automatically

1D Automatic Peak Picking

Since peak picking is a crucial part of the spectral assignment process, SpecMan provides 1D and 2D auto peak picking capabilities (SpecMan+ provides 1D, 2D and 3D peak picking capabilities). During the peak picking procedure, SpecMan searches for true peak tops by using a sophisticated matrix search procedure. Only those peaks whose intensities are above the specified noise threshold are reported, and peaks identified through this process are displayed in a peaks table.

When ‘Pick Peaks Automatically – 1D’ is selected from the Display menu, the following dialog box appears:

The dialog box displays the current spectral information and prompts the user to enter peak types (positive, negative, or both), noise threshold, search limits, and a toggle to control whether to overwrite or append to the existing peaks table. In general, the threshold and zoom limits will have been set appropriately before initiating the peak picking process. Check the other boxes appropriately and then click ‘OK’ to perform 1D auto peak picking. Once the peaks have been picked, the peaks will be annotated with a peak label, and the 1D peaks table will be displayed with the picked peaks.

Click ‘OK’ to perform 1D auto peak picking. Once the peaks have been picked, the peaks will be annotated with a peak label, and the 1D peaks table will be displayed with the picked peaks.

2D Automatic Peak Picking

SpecMan provides a variety of 2D peak picking methods for efficient spectral data abstraction. SpecMan offers the user four types of peak picking procedures to handle different types of 2D NMR, 2D HPLC-MS and 2D HPLC-UV data. These methods include advanced noise filtering and intelligent peak identification techniques. The four types of peak picking procedures are:

Standard Peak Picking

During the standard peak picking procedure, SpecMan searches for the true peak tops using a sophisticated matrix search procedure. Only those peaks whose intensities are above the specified noise threshold are reported and picked. This method works well on well-resolved peaks, but it cannot be used for picking cross peaks that have multiplicity.

When ‘Pick Peaks Automatically – 2D’ is selected from the Display menu, the following dialog box appears:

For standard peak picking, the following options are used: peak type filter (positive, negative or both), diagonal filter (if it is a homonuclear 2D data), threshold and clear current peaks table.

Multiplet Merging

Auto peak picking and merging of peak multiplets is used for peak picking the center of mass of cross peaks with multiplets (for example HMQC, DQFCOSY, etc). This method requires two more selections in addition to the options described above for standard peak picking. The first additional feature is merge peak multiplets, and is used to determine the process of merging peak locations as they get picked. The second option is for discriminating noise peaks from real peaks.

The ‘Merge Peak Mulitplets’ option can be based either on Average, Weighted Average or Highest Peak. When Average is selected, the shifts of multiplets will be averaged to determine the shift of the center of mass. When Weighted Average is selected, the shifts are weighted with the intensities of the multiplets, and when Highest Peak is selected, only the shift of the multiplet that has the highest intensity is retained as the shift of the center of mass. The weighted average option is the default, and for most cases, the weighted average option works the best.

The ‘Peak Width Filter’ option discriminates noise peaks from real peaks by prompting the user to enter minimum and maximum values for peak width filters. These minimum and maximum values define a rectangular-sized box that identifies the dimensions of a cross peak. The minimum X and minimum Y values (in ppm’s) filter out noise peaks, because all peaks that are smaller than the dimension of the box defined by these limits are treated as noise peaks and eliminated from the peak list. The maximum X and Maximum Y (in ppm’s) are used to define the upper limit for the size of a cross peak. SpecMan uses a rectangular box defined by these limits to determine the grouping of multiplets within a cross peak. If all the multiplets lie within this limit, then they are assumed to be part of the same cross peak and the multiplet picking option (average, weighted average or highest peak) is applied to retain the location of the cross peak. If the user prefers to set these limits graphically, the user can select the ‘Set Graphically’ button for Peak Width Filter. This will momentarily close the ‘Pick 2D Peaks’ dialog box so that the user can draw a rectangular box (with the left mouse button) around a cross peak for setting the maximum limits, and the user can draw a rectangular box around a weak noise peak to set the minimum limits. Note that the user can also leave the minimum limits as 0.00, but if there is a strong t1 ridge, the user will want to filter out the weak noise peaks. This method is beneficial when the data contains peak multiplets.

Grid Intelligence Based Peak Picking

Auto peak picking with grid intelligence uses 1D reference peak lists as a guide to locate potential 2D peaks. This mode works well with NOESY and TOCSY data sets in which the user wants to filter out t1/t2 ridges. Prior to auto peak picking using grid intelligence, it is necessary to associate a 1D peak list with the 2D spectrum (please refer to the ‘Associate Reference Spectra’ section of this guide for details). Once the 1D peak lists have been associated, select ‘Pick Peaks Automatically – 2D’ from the Display menu and check ‘Grid Intelligence’ from the 2D peak picking dialog box (uncheck the ‘Merge Peak Multiplets’ option).

Grid intelligence requires minimum X and minimum Y tolerances. These values are used to define the distance cut-off for filtering peaks that are not within a specified distance form a 1D peak list grid intersection. SpecMan will ignore all peaks that fall outside of this specified distance. If the user prefers to set these limits graphically, the user can select the ‘Set Graphically’ button for Grid Distance Filter. This will momentarily close the ‘Pick 2D Peaks’ dialog box so that the user can draw a rectangular box (with the left mouse button) around a noise peak to set the minimum X and Y limits. This method is beneficial because only peaks that are very close to the grid intersection are retained, and this helps in filtering out the majority of the noise in the t1/t2 ridges. In addition, real peaks will be retained, even if they happen to lie on t1/t2 ridges. However, since this option doesn’t merge peak multiplets, this procedure is useful only for picking peaks that are pure singlets.

Grid Intelligence Based Peak Multiplet Merging

This method is a combination of Multiplet Merging and Grid Intelligence Based Peak Picking. To use this option, select both ‘Merge Peak Multiplets’ and ‘Grid Intelligence’ from the Pick 2D Peaks dialog box as shown below:

The utilization of four peak width filters (Minimum X & Y and Maximum X & Y) combines the grid intelligence use of the Minimum X and Y values to determine minimum distance from grid lines, and peak merging use of the Maximum X and Y values to determine the box size for the merging of peaks. The ‘weighted average’ multiplet option is used in the same manner as it was described above in multiplet merging. To find appropriate peak width filter values, it is recommended that the user zoom in on different regions of the spectrum to experiment with different values. By measuring the differences in PPM along X and Y around a noise peak and a real cross peak, the user can determine the appropriate limits for the peak width filters. Once the optimal peak width filters have been determined, click ‘OK’ and SpecMan will perform peak picking.

It is important to point out that grid intelligence peak picking can even be used when there is only one associated 1D peak list (i.e. peaks along either X or Y). For instance, before extracting a 1D proton peak list from 2D HMQC, the user can perform grid intelligence-based peak multiplet merging using only the associated 1D Carbon peak list on the Y axis.

When analyzing other spectral data, the following dialog boxes (Mass Spec and UV, respectively) will be displayed:

3D/4D Automatic Peak Picking

In SpecMan+, 3D peak picking is based on similar methods as in 2D, except that during the search procedure the points in the third dimension (or tier planes) are also included for locating peak tops. When the extrema lie between two tier planes the chemical shifts will be interpolated. The 3D peak search procedures can also be combined with 2D grid intelligence and auto peak merge methods as applied in 2D auto peak picking. Automatic 4D peak picking can be performed by doing a series of 3D auto peak picking of individual 3D bricks in a 4D spectrum.

6.3 Add Peaks Manually

SpecMan enables the user to add peaks manually (appended to a new or existing peaks table) either before or after automatic peak picking. The following options are available from the pullright menu of ‘Add Peaks Manually’ from the Analysis menu:

· Singlets

· Multiplets

· Without Refine

· Grid Location

Singlets

The ‘Singlets’ option permits the user to add peaks at locations that SpecMan considers the proper location for the maximum peak intensity. Once the ‘Singlets’ option has been selected, click on a peak in the spectrum, and SpecMan will search the spectral data for a local maximum (or minimum for negative peaks) and label the peak.

Note: SpecMan will warn the user if the user attempts to add a peak top that has already been picked and labeled, or if the intensity of a peak location is lower than the current threshold. The user can either lower the threshold or select ‘Add Peaks Manually - Without Refine’ instead.

Multiplets

The ‘Multiplets’ option allows the user to add a single peak from multiple peak tops (useful when picking COSY peaks). Once the ‘Multiplets’ option has been selected, define a multiplet region (using the rubberband zoom). SpecMan will search this region for peak tops, and compute the location for the peak based on the current ‘Peak Picking Options’ (average, weighted average, or highest peak).

Note: SpecMan will warn the user if no peaks are found within the specified region. The user should either lower the threshold, or use ‘Add Peaks Manually Without Refine.’

Without Refine

The ‘Without Refine’ option is used to add peaks at any user-specified location. Once the ‘Without Refine’ option has been selected, SpecMan will add a peak at the exact location where the user clicks (and not search to see if the specified location is actually a local maximum or minimum).

Note: SpecMan will warn the user if the user clicks close to an already existing peak, and the user will have to select a different location.

Grid Location

The ‘Grid Location’ option is used to add peaks at a gridline intersection. When adding gridline peaks, 1D peak lists must first be associated along the X and Y dimensions of the 2D spectrum. Once the ‘Grid Location’ option has been selected, click on a location and SpecMan will add a peak to the closest grid intersection.

Warning: This option can’t be used with 1D spectra, since 1D spectra don’t have associated peak lists.

6.4 Modify Grid

After associating a peak list with a spectrum, the user may find that some of the 1D peaks were picked in the wrong location, picked in error, or missed entirely. Rather than re-opening the 1D spectrum and its peak list, and then editing the list, SpecMan allows the user to add and remove associated gridlines. This feature, in turn, edits the underlying peak list file.

The following pullright options are available from ‘Modify Grid’ on the Analysis menu:

Add X Gridline

Add Y Gridline

Remove X Gridline

Remove Y Gridline

To modify the associated peak list, select the appropriate pullright option and then click on a desired location to add a gridline at that position, or click on an existing gridline to remove it. Additional selection of the option will turn the feature off.

Note: To move a gridline, the user must first delete the line, and then add a new line in the proper location.

Hint: The user can switch to a different ‘Modify Grid’ option without deactivating the old one first. Simply select the new option, and the old option will be deactivated automatically.

6.5 Merge Peaks

SpecMan allows the user to combine several peaks into one peak. Select ‘Merge Peaks’ from the Analysis menu and use the mouse to select the region to merge peaks within by clicking, dragging and releasing the rubber-band box. All peaks located within the selected region will be merged into one peak.

Note: The method used to place the merged peak is determined by the option selected under the ‘Peak Picking Options’ pullright menu in the Analysis menu.

6.6 Remove Peaks

SpecMan enables the user to remove peaks picked through auto and manual peak picking procedures (useful in eliminating peaks that arise from noise and other artifacts in the spectrum). This cleaning process deletes peak entries in the peaks table and the deleted peaks will no longer appear annotated in the spectral window.

When ‘Remove Peaks’ is selected from the Analysis menu, SpecMan prompts the user to select a region (by using a rubber-band zoom) in which all peaks will be removed. When the rubber-band zoom has been completed, all peaks in the enclosed region will be deleted from the table, and the labels of these peaks will no longer appear in the spectral window.

Peaks can also be removed by deleting the peak entry from the Peaks Table. To do so, highlight a peak entry and then click the ‘Delete’ key on the keyboard.

Note: The undo feature is not available for ‘Remove Peaks.’ If a good peak has been deleted by mistake, the user will have to add it again by selecting ‘Add Peaks’ from the Analysis menu.

6.7 Move Peaks

After picking a peak manually or automatically, the user may decide that the peak location needs to be shifted slightly. Select ‘Move Peaks’ from the Analysis menu and click on a peak symbol (‘+’) and drag to a new location. The peaks table will be updated to reflect the new peak location. Reselect ‘Move Peaks’ again to deactivate the feature.

6.8 Show Peaks

SpecMan enables the user to display or hide the peak labels of peaks picked by auto or manual peak picking. Peaks are annotated with a ‘+’ symbol at the precise location of the peak, and are labeled with the character string specified in the label column of the peak table (the peak ID number, by default). Select ‘Show Peaks’ from the Analysis menu to display the peak annotations and reselect the option to deactivate the feature.

6.9 Annotate Peaks

SpecMan allows the user to manually annotate peaks by selecting ‘Annotate Peaks’ from the Analysis menu. The user will be prompted to click on the intended peak, and once the peak has been clicked on, the following ‘Change Peak Label’ dialog box will appear:

In this dialog box, the user can type in numbers and also select Greek letters (or capital letters) from the dialog box. Click ‘OK’ to accept the new label for display in the spectral window.

6.10 Modify Peaks

This function allows the user to define the Multiplicity and the J Coupling values of spectral peaks, in addition to Manual 1D and 2D Integration. The following options are available from the pullright menu of ‘Modify Peaks’ from the Analysis menu:

Multiplicity

J Coupling

Manual 1D Integration

Manual 2D Integration

Multiplicity

During automatic peak picking, the multiplicity values by default are listed as ‘u’ for ‘unknown,’ in the 1D Peaks Table as shown below:

In order to manually define multiplicity values, select ‘Modify Peaks’ from the Display menu, with a pull-right menu selection of ‘Multiplicity,’ as shown below:

By selecting ‘Multiplicity’ the following dialog box will appear:

Select which type of multiplicity to assign (for example, ‘s’) and then select OK. Next, click on the correct entry line in the Peaks Table, and the multiplicity will be changed from ‘u’ to ‘s’ as shown in the following:

The corresponding peak in the spectrum will be highlighted to indicate that its multiplicity has been modified. Continue to click on additional entries in the peaks table to define more ‘s’ multiplicity values, or select a different type of multiplicity to define from the Edit Peak Multiplicities dialog box. Once the user is finished defining multiplicity values, select Cancel from the Edit Peak Multiplicities dialog box to turn off this feature.

Note that NMR-SAMS automatically assigns multiplicity for Carbon data when Carbon, DEPT 45, DEPT 90 and DEPT 135 peak lists are available during its structure elucidation process.

J Coupling values are measured in Hertz, and by default are listed as zero in the 2D Peaks Table:

In order to manually define J Coupling values, select Modify Peaks from the Display menu, with a pull-right menu selection of J Coupling, and the following dialog box will appear:

Type in the J Coupling value to define (for example, 5), and select OK. Next, click on the correct entry line in the Peaks Table, and the J value will be changed from 0.00 to 5 as shown below:

The corresponding peak in the spectrum will be highlighted to indicate that it’s J Coupling value has been modified. Continue to click on additional entries in the peaks table to define more J coupling values of 5, or select a different value to define from the Edit J Couplings dialog box. Once the user has finished defining J coupling values, select Cancel from the Edit J Couplings dialog box to turn off this feature. This feature is still in development, and future additions will improve its flexibility to define additional J Coupling values.

Manual 1D Peak Integration & 2D Peak Volume Computation

SpecMan enables the user to perform manual 1D integration once peak picking has been performed. By selecting ‘Manual 1D Integration’ from the ‘Modify Peaks’ pull-right menu, the user will be prompted to define an the integration area of the peak by clicking and dragging the left mouse button as shown below:

When the left mouse button is released, the following ‘Calibrate Integral’ dialog box will appear:

Click ‘OK’ and the peak will be integrated as 1 Proton, as shown below:

To integrate other peaks using the first peak as the standard, define a rubber-band zoom box around the next peak:

When the left mouse button is released, the 2^nd peak will be integrated as shown below:

The integration value of each peak is listed in the Peaks Table as shown below:

In a similar manner, the user can perform manual 2D peak volume computation once peak picking has been performed. The following ‘Calibrate Integral’ dialog box will appear after an integration area has been defined around a 2D peak:

Click ‘OK’ and the 1^st peak (along with the 2^nd peak) will be integrated as shown below:

6.11 Relocate Peak Labels

This option allows the user to relocate peak labels of picked peaks. Select ‘Relocate Peak Labels’ from the Analysis menu as shown below:

Then, click on a peak label and drag the label to a new position as shown below:

6.12 Manual Assignment

This function is used for manual assignment of chemical shifts. By selecting Manual Assignment from the Analysis menu, the following pull-right options will appear:

To utilize this function, open up a spectrum with its corresponding 1D or 2D peaks table, along with a molecule. Then, open up a blank assignment table by selecting Assignment Table from the Display Menu. Next, select Manual Assignment from the Analysis menu with pull-right menu of C-13 to manually assign the Carbon chemical shifts. Select the desired chemical shift value from the peaks table (for example, 151.32), and then click on the corresponding atom in the molecule (for example, 2) to manually assign its chemical shift as shown below:

Atom 2 now has a defined chemical shift of 151.32, and this number gets updated in both the molecule and the assignment table. The user can also click on a peak in the spectrum (rather than an entry in the peaks table), and have the chemical shift from the chosen peak assigned to the atom in the molecule. In a similar manner, the proton chemical shifts can also be manually assigned.

After manual assignment is completed, it is important to select ‘Save’ from the Assignment Table in order to save any changes to the assignments. By selecting ‘Save’, the assignment values are appended to the end of the structure files (either *.mol or *.mdl). This ensures that when the corresponding molecule is reopened, the modified assignment values will also be included. Please note that SpecMan will only allow an Assignment Table to be loaded as long as it matches the current active molecule.

6.13 Crosshair Cursor

This feature gives the user the option to display or hide the crosshair cursor. The cursor is displayed by selecting ‘Crosshair Cursor’ from the Analysis menu and selection again will deactivate the crosshair cursor (the cursor will appear as an arrow).

With the crosshair cursor on, the user can:

· Display the X & Y axes locations on the status bar.

· Determine the intensity value of a peak in a 2D spectrum by double-clicking on the peak.

· If the 1D Slice option is selected and the slice windows are open, the current location of the cursor is displayed in the slice view status bar.

· Select new 1D slices of a 2D spectrum by clicking on a peak in a 2D spectrum.

· Freeze the current location by clicking the right mouse button.

The color of the crosshair cursor can be changed in the specmanpersonal.ini file.

Chapter 7

SpecMan’s Help Menu

The Help menu lists the options related to the online help of SpecMan. This chapter helps the user navigate SpecMan’s Help Menu and its features, as well as relevant main concepts and principles.

7.1 Tutorial

SpecMan offers the user program assistance in the form of online help. By selecting ‘Tutorial’ from the Help menu, the following will appear:

Click on either ‘Q-2’ or ‘Paclitaxel’ to view a tutorial using Varian data (Q-2) or Bruker data (Paclitaxel).

In order to be able to view the online help and tutorials in SpecMan, the user needs to configure SpecMan’s *.ini files so that the online help uses the user’s internet browser (either Netscape or Internet Explorer). To do so, modify the following section in the *.ini files:

# If you are running Windows 95/98/2000, use the following set-up for your web browser: # Netscape is the default web browser. If you are using a different browser, # comment the Netscape line below, and un-comment the Internet Explorer line below: WebBrowser C:\Program Files\Netscape\Communicator\Program\netscape.exe # WebBrowser C:\Program Files\Internet Explorer\Iexplore.exe # # If you are running Windows NT 4.x, use the following set-up for your web browser, since # NT is sensitive to any spaces in filenames. Therefore, use the shortened DOS equivalent as # shown below: # Netscape is the default web browser. If you are using Netscape, uncomment the # Netscape line below, but if you are using Internet Explorer, uncomment the Internet # Explorer line below: # WebBrowser C:\Progra~1\Netscape\Communicator\Program\netscape.exe # WebBrowser C:\Progra~1\Plus!\Micros~1\Iexplore.exe

The above is a sample pathname; the user needs to put in the actual pathname for their internet browser (either Netscape or Internet Explorer).

7.2 Contents

SpecMan enables the user to view sections of this user guide inside the SpecMan program. By selecting ‘Contents’ from the Help menu, the following will appear:

The user can then scroll through the help topics and select the necessary topic and a page will appear containing help information.

7.3 About SpecMan

The option ‘About SpecMan’ lists the version of SpecMan, the date the version was released, a basic description of the SpecMan program and contact information for Spectrum Research, LLC.

Index

<Folder>/File	Required/Optional	Purpose
specman.exe	Required	Primary Executable
license.dat	Required	Licensing Information
specman.ini	Required	User Customization File
specmanpersonal.ini	Required	User Customization File
specmanblack.ini	Required	User Customization File
specmanwhite.ini	Required	User Customization File
ps.defaults	Required	Defaults for postscript output
specman.cnt	Required	Help contents
periodic_tab.def	Required	Periodic Table of chemical elements
rgb.txt	Required	Color definitions
<Help>	Required	Online Help directory
<data>	Optional	Sample Spectral Data