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CHAPTER 4: Tutorial II: Setting Analysis Parameters

In CHAPTER 3: "Tutorial I: Using NMRanalyst", input parameters for the analysis of the prednisone dataset are provided. For new datasets, NMRanalyst contains default settings for the supported spectrum types. A load function is provided to add Bruker and Varian FID and spectra specific parameters to these generic settings. So the number of parameters which may need optimization is limited. The procedure to optimize them is described in this tutorial.

4.1 The UNIX Shell Window

If an NMRanalyst application is still running, exit it to remove it with its started plot applications. Now start NMRanalyst and change to the supplied data directory as described in the previous tutorial (CHAPTER 3: "Tutorial I: Using NMRanalyst"). For the analysis of a new dataset, no customized input value files are available. Select from the Spectrum Type menu [1,1-ADEQUATE].

Click the [UNIX Shell] button at the top of the NMRanalyst window. A shell window similar to the shown one appears. It can execute UNIX commands, even on Microsoft Windows. Rename the INADEQUATE directory containing current parameter values from this shell window:

 % mv INADEQUATE INADEQUATE.org

When no spectrum specific directory is present, NMRanalyst loads generic files from the $NMRUSER/INADEQUATE directory. Now change NMRanalyst back to the INADEQUATE spectrum type: Spectrum Type menu [INADEQUATE] item.

4.2 1D Analysis Workwindow Settings for Prednisone

Click the [1D Analysis] tab in the NMRanalyst window. As no customized settings are available, NMRanalyst provides default settings. Unknown spectrum related fields such as Observe Frequency, Spectral Width, and Start of Spectrum remain empty. Click the [Load] button at the top of the application window. In the displayed Filebox popup, double click in the Folders list on the left the directory named 5, and double click in the Files list on the right file acqus containing the spectral parameters. This load process is also described as "load from directory 5 file acqus" or simply "load 5/acqus".

The spectrum specific parameters are loaded by this procedure. Click [Start]. This operation is also described as running the workwindow. Twenty-eight 1D resonances are reported. The 1D Analysis workwindow saves the resulting phase corrected experimental and simulated spectra in file carbon.plot.

Switch to the Graphic workwindow (click the NMRanalyst window [Graphic] tab). Set the File With Plot Data input field to carbon.plot by either typing the new name, or by clicking on the folder button at the right side and choosing this file from the started Filebox. Run the workwindow to display the shown overlaid spectra.

In the displayed plot, the experimental spectrum is drawn in yellow first and then the simulated spectrum is drawn on top in black. No resonance of significance should remain in yellow, as this would imply it was not properly identified by the automated analysis. Small differences between detected and simulated resonances result from magnet inhomogeneity (imperfect shimming) and spectral noise. Here the spectral baseline is sufficiently flat and all resonances are identified.

The seven DMSO-d6 solvent resonances in this spectrum are around 40 ppm: Place the cursor at the top left edge of the area of interest in this spectral plot, drag the cursor with pressed left mouse button and a yellow "rubber-band" indicates the currently selected region. Release the mouse button and the selected area redraws enlarged. If the area is still too large, zoom in again. If the area is not correct, select [Reset] from the [View] pull-down menu at the top of this window to undo all zooming or select [Previous Zoom] to undo the last one. The shown enlarged spectral area shows the DMSO-d6 solvent center resonance around 40.3, while it should be at 39.5 ppm. Display the 1D Analysis workwindow. Click the [Output Screen] button to display the analysis results again. At the bottom of the output screen, NMRanalyst identifies the DMSO-d6 solvent resonances and the spectral referencing is -109.960 Hz off from the expected referencing:

 DMSO Resonances: 16 17 18 19 20 21 22  Start of Spectrum -109.960 = 1753.281 Hz

To load the determined resonance descriptions with reference correction in the 1D Analysis input screen, load from directory INADEQUATE the file 1d_analysis.log (click [Load] and select INADEQUATE/1d_analysis.log). Scroll to the bottom of the 1D Analysis input screen in which the numerical description of the determined 29 carbon resonances is loaded. The removal of the seven solvent resonances is already requested by selected Remove check buttons for each solvent resonance, as shown.

Rerun the 1D spectral analysis. Display the resulting carbon.plot file from the Graphic workwindow as before. Confirm the seven DMSO-d6 resonances are removed except for minor residuals which are identical to the ones observed before removing these resonances.

4.3 VerifyIt: Molecular Structure Verification and Shift Assignments

SECTION 3.1: "findit Script for Molecular Structure Identification" covers FindIt, the determination of best matching structures for NMR data. Often the likely structure of a molecule is known and only needs to be validated by NMR. Perhaps the molecule was synthesized by well established reactions and only its identity needs to be confirmed. Each NMRanalyst supplied sample dataset directory contains the Molfile of the examined molecular structure. From the Graphic workwindow, display the shown 4900.mol prednisone structure. (Bonded hydrogen atoms are implied and not explicitly displayed.)

Click the [AssembleIt] tab. Select the [VerifyIt: RATE SPECIFIED STRUCTURE BY AGREEMENT WITH NMR DATA] switch. Set its Proposed Structure to 4900.mol and Assigned Carbon Shifts to 4900.plot. Run the workwindow. VerifyIt shows a detailed comparison of the detected 1D carbon results with the expected molecular structure. The overall prednisone rating is 0.993954 on a scale of 1.0 for perfect agreement and 0.0 for no agreement. Compared to over 15.9 million FindIt structures of small organic molecules, this specified structure obtains the top position for the observed NMR data.

To derive this structure rating, VerifyIt assigns observed carbon shifts to the specified structure. This assigned structure is saved in the specified 4900.plot file. Display it from the Graphic workwindow as shown in the top figure. For the following shift prediction, select in the NMRgraph window [Edit][Preferences...]. In the popup set Shift Prediction Method: to [HOSE-Code Before Rule-Based] and click [OK]. To compare observed with predicted shifts, select [Prediction] [13C Shifts]. To display the shift differences, select [View] [Show 13C Shift Differences] as shown in the bottom figure. Differences up to 5 ppm represent a reasonable agreement for carbons. For shifts closer than 5 ppm, exchanged assignments might result.

4.4 FFT Workwindow Settings for Prednisone

Switch to the FFT workwindow. Like the 1D Analysis workwindow before, it initially only contains default INADEQUATE parameters. Load the spectrum specific parameters by loading from the directory named 7 the file acqus. It is important to keep the referencing of all spectra consistent. To bring the major DMSO-d6 solvent resonance to 39.5 ppm, the 1D carbon spectrum Start of Spectrum is set to 1754.059 in the 1D Analysis workwindow. The 2D INADEQUATE spectrum solvent is specified as CDCl3. But the spectrum shows no CDCl3, but DMSO-d6 resonances. So also set F2: Start of Spectrum to 1754.059. Run the workwindow.

4.5 Displaying All Workwindow Input Fields

For the routine use of the software, many input fields are not needed and are not displayed. Select from NMRanalyst the Edit menu [Preferences...] popup. Select the [Show All Input Fields] switch and click [OK]. Now all settings of the software are displayed.

Only the completed 1D Analysis and FFT workwindows require additional acquisition specific information to be loaded. Switch to the 2D Analysis workwindow. At the bottom of its input screen the [RUN REPORT WORKWINDOW AFTER SUCCESSFUL ANALYSIS] switch is shown. This switch is selected by default, whether it is displayed or not. Run the workwindow. As soon as the spectral analysis completes, NMRanalyst switches to the Report workwindow and runs it to summarize the analysis results.

With the 2D analysis default settings, all but one prednisone carbon-carbon bond are detected. The 49.78 to 50.45 ppm carbon bond is highly second order and is not detected well by the used pulse sequence. The following refinement will detect this bond.

4.6 Determining Phase Functions

For the visual NMR data analysis, phase sensitive spectra are phase corrected. This improves the resolution of the displayed spectrum by removing dispersive signal components. Absorption resonances have a higher amplitude and are easier to distinguish visually from noise. NMRanalyst does not phase correct data for the numerical analysis, as it analyzes all phase components, two for 1D, four for 2D, and eight for 3D phase sensitive spectra. Phasing merely redistributes information between the analyzed phase components and frequency dependent phasing distorts line shapes. But NMRanalyst automatically determines phase functions. They are used to display experimental data phase corrected. They are also used so that NMRanalyst does not have to redetermine the phase of every spin system in each dimension. This improves analysis speed and sensitivity.

NMRanalyst determines the phase of every 2D INADEQUATE correlation for both spectral dimensions. Determined phases can be plotted against their spectral frequencies. Interpolation of these phases yields the F1 and F2 phase functions. NMRanalyst determined them during the last Report workwindow run. Switch to the Graphic workwindow input screen, click on the File With Plot Data folder button on the right, double click on the file phases.plot, and click [Start] to display the shown plot. Phases are given on the vertical axis in radians (to convert them to degrees, multiply them by 180/) and the marginal standard deviation in phase and frequency is shown as one tilted error bar through each phase value. This phasing works so well, we patented it.1 For comparing determined phase functions, remember that a phase value only influences the appearance of the NMR spectrum through the sine and cosine of its value. So a phase function shifted by 2 is identical to the unshifted one. For higher dimensional spectra, simultaneously shifting the phase in two orthogonal dimensions by does not alter the spectral phasing either.

To use these phase functions for the correction of spectral plots, load in the Graphic workwindow from directory INADEQUATE the file report.log. Also load these functions in the 2D Analysis workwindow to improve the spectral analysis. Now in the 2D Analysis input screen both linear phase functions are set and the Mapping: and Detection: [F1 Phase] and [F2 Phase] switches are deselected as shown. Run the 2D Analysis which auto-runs the Report workwindow.

4.7 Prednisone Spectrum With 1D Carbon & Correlation Locations

Switch to the Graphic workwindow input screen. In the File With Plot Data input field specify inadequate.spec, the spectrum created by the FFT workwindow. Run the workwindow to display the shown spectrum plot. It shows the phased experimental prednisone 2D INADEQUATE spectrum with the 1D carbon spectrum. The location of NMRanalyst identified correlations are indicated by rectangular bounding boxes. The spectral F1 sweep width (vertical axis) was acquired with equal width as the F2 axis. Had this spectrum been acquired with a larger F1 spectral range, the green bounding boxes of aliased resonances would have been detected outside the shown spectrum. The unaliased correlations are indicated by black bounding boxes.

4.8 Experimental, Simulated, and Residual Spectrum of One Correlation

Detailed plots are only created when requested. To identify the weakest detected prednisone correlation, switch to the Report workwindow and click [Output Screen]. Scroll up to the Correlation Detection Probabilities table. The weakest bond is at the bottom of this table:

 Index     P(I)      Prob.                 C1   C2    Fa        Fb         J   
 ------------------------------------------------------------------------------
 #     7   2.747  0.9999912  CORRELATION    1    8   211.416    87.402    46.29

Switch to the 2D Analysis workwindow. In the input field Analyze Correlation Index #s specify the index number 7. Scroll to the bottom of the input screen and select the [Combined Experimental, Simulated, Residual Spectrum] check button, deselect the [RUN REPORT WORKWINDOW AFTER SUCCESSFUL ANALYSIS] check button, and run the workwindow. The creation of file Fa211.42_Fb87.40_J46.3.plot is reported.

Switch to the input screen. Delete the Analyze Correlation Index #s entry again. Deselect the [Combined Experimental, Simulated, Residual Spectrum] switch and select the [RUN REPORT WORKWINDOW AFTER SUCCESSFUL ANALYSIS] switch again. Change to the Graphic workwindow, set File With Plot Data to Fa211.42_Fb87.40_J46.3.plot, and click [Start]. The 87.40 ppm anti-phase doublet on the right side is observable. But the 211.42 ppm anti-phase part of this bond on the left side requires this software's three times higher sensitivity over the visual analysis for detection.

Display file structure.plot from the Graphic workwindow. The complete prednisone carbon skeleton is now determined.

4.9 The History Mechanism for Input Values

Every input field in NMRanalyst keeps a history of previous entries. Display the Graphic workwindow input screen and click on the File With Plot Data button. The displayed popup is shown. This capability can be used as an undo facility. Furthermore, selecting previous values from the history list is more convenient than typing them in again. The current value is the bottom entry in this list and the default value is the first entry in the list. For an empty input field the history list entry is "(empty)". Double click now on structure.plot to copy it in the input field. The background color of this input field changes to the default color.

To make current input screen settings the default values, click the [Workwindow] pull-down menu and select [Make Default]. This mechanism is helpful to declare the current settings as the starting point before further alterations which are then easily visible as "non-default" values.

4.10 Directory Editor and the Sucrose FID Analysis

The 8.2 mg sucrose (MF: C12H22O11, D2O solvent) 2D INADEQUATE dataset was acquired overnight on a Varian UNITYplus 500 spectrometer using a 13C Nanoprobe.2 Start NMRanalyst. This sucrose_INADEQUATE dataset is provided with the NMRanalyst software. Assuming it was installed in the UNIX system directory /usr/local and should be copied to the login directory, start the UNIX Shell, and issue the commands:

 % cp -r /usr/local/sucrose_INADEQUATE $HOME
 % cd $HOME/sucrose_INADEQUATE

Otherwise load it from the original NMRanalyst distribution medium (e.g., a DVD). Rename the contained INADEQUATE directory, so the provided analysis parameters are not found (still from the UNIX Shell):

 % mv INADEQUATE INADEQUATE.org

Click the [Directory Editor] button at the top of the NMRanalyst window to display the shown popup. All NMRanalyst directories can be set from this popup. Set the current working directory NMRDATA to $HOME/sucrose_INADEQUATE. Set its spectrum type NMRSPEC to INADEQUATE, and click [OK].

Use the procedure described for prednisone. Only small adjustments are needed for this sucrose dataset:

  1. In the 1D Analysis workwindow [Load] Filebox, select for this Varian format dataset directory 1d.fid and then file procpar.
  2. In the FFT workwindow [Load] Filebox, select directory 2d.fid and then file procpar. For Varian format FID data, the directory name always ends on .fid and the file with acquisition parameters is always called procpar.
  3. The sucrose sample does not contain a referencing compound and its D2O solvent contains no carbons. So no solvent resonances need to be removed in the 1D Analysis workwindow.

The determined sucrose carbon skeleton is shown. A 2D INADEQUATE spectrum does not detect carbon-oxygen bonds and two carbon fragments result. To reproduce stated shifts and coupling constants, make sure the report.log file is loaded in the 2D Analysis workwindow, to set and lock the F1 and F2 phase functions.

4.11 Analysis of the Sucrose VNMR Created Spectrum

Besides transforming FIDs, NMRanalyst can import and analyze phase sensitive spectra created by Varian (e.g., VNMR) and Bruker (e.g., XWIN-NMR) software. Incentives to analyze externally created spectra include:

From the previous section, the sucrose NMRanalyst parameters are configured to analyze FIDs. The Varian VNMR software stores transformed spectra with all its phase components in file ~vnmr1/vnmrsys/exp?/datdir/data (the question mark represents a number between 1 and 9). The data files are provided in the sucrose_INADEQUATE subdirectories 1d.fid and 2d.fid. Change the 1D Analysis workwindow 1D FID, Spectrum, or Line List field to 1d.fid/data and click [Start]. Change the FFT workwindow 2D FID or Spectrum field to 2d.fid/data and click [Start]. Run the 2D Analysis workwindow which auto-runs the Report workwindow. From the Graphic workwindow display the carbon skeleton. It is the same as the one from the previous section except for minor coupling constant deviations.

As in SECTION 4.3: "VerifyIt: Molecular Structure Verification and Shift Assignments", create the shown carbon assignments for the 1115.mol sucrose structure. VerifyIt only assigns the 1D carbon spectrum results to this structure. It agrees with the 2D INADEQUATE detected carbon-carbon skeleton.

1U.S. Patents 5,218,299 and 5,572,125, British Patent 0 577 770, German Patent 692 31 690.6-08.

2Courtesy of Paul A. Keifer, Ph.D., Varian Inc.



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