This chapter describes the Report workwindow summarizing the automated two- and three-dimensional spectral analysis results. Its input fields and resulting program output are explained. (To display all the input fields available in this workwindow, select from the
Mode: [Full NMRanalyst],
[Show All Input Fields] switches, and click
[OK].) See CHAPTER 12: "Using the Workwindows" for a general description of the function and use of a workwindow.
report, controlled by the Report workwindow, assembles the information obtained from the 1D Analysis, FFT, and nD Analysis workwindows for the selected multidimensional spectrum type. The
analyze program, controlled by the nD Analysis workwindow, examines one fitting area at a time and does not attempt to assemble the analysis results from different fitting areas. The program
report summarizes this information and provides warning messages in case of ambiguous signal assignments, a phase mismatch of a reported correlation with the overall phase functions, or overlapping correlation areas.
The AssembleIt workwindow can combine the Report workwindow results from different spectrum types. Its FindIt component identifies the best matching molecular structures for the observed NMR data. Its AssembleIt component derives the most likely skeletons from this often incomplete and inconsistent correlation information, see CHAPTER 17: "AssembleIt Workwindow" for details.
Determined ?D Correlationsfield. The file contains the numerical description of identified patterns in the analyzed multidimensional spectrum. (Key:
For 2D INADEQUATE spectra, the resulting graph shows the determined carbon skeleton. For heteronuclear spectra, the displayed fragments consist of one carbon atom with correlated proton frequencies. For NOESY, ROESY, and TOCSY spectra, each displayed fragment contains a proton surrounded by correlated proton frequencies. The AssembleIt workwindow allows combining the information from different spectrum types and deriving the possible carbon skeleton(s) from it. (Key:
Specify a name for the plot file of determined phase values and phase functions. This plot shows the precision with which the
report program determines the spectral
Phase Functions. The plot file is created when a file name is specified in this field. Use the Graphic workwindow to display it. (Key:
The Report workwindow produces a connectivity table and warnings. This output file contains a plot of areas of 2D or volumes of 3D spectrum showing the location of determined correlation signals. The plot file is created when a file name is specified in this input field. Use the Graphic workwindow to display it. (Key:
The normal (Gaussian) distribution of parameter precisions or agreements of non-correlation patterns allows calculating correlation detection probabilities. Specify a name in this input field to save a plot file of the distribution of analyzed correlation areas, the threshold for detected correlations, and the calculated distribution function. Use the Graphic workwindow to display it. (Key:
Precision Distribution, described above, allows effective distinction of signals from random noise. But spectral distortions, especially T1 ridges, are not random and often consist of signals which match the spin system model. Specify a filename in this input field to save the integral distribution and use the Graphic workwindow to display it. NMRanalyst determined integrals correspond to the integrals of well-phased signals. Based on this integral distribution plot, choose an integral value threshold above the noise and ridge signal responses. To exclude the weak signals, specify this threshold in the nD Analysis workwindow
Thresholds: Integral field as described in CHAPTER 15: "nD Analysis Workwindow" and rerun the analysis. (Key:
Acquiring a 1D carbon spectrum is 5700 times less sensitive than a 1D proton spectrum. A 1D nitrogen spectrum is 50 times worse than carbon. Acquisition of a 1D carbon spectrum often takes as much time as a multiplicity edited HSQC, HMBC, and DQF-COSY spectra combined. The acquisition of a nitrogen spectrum is impractical. Equivalent resonance information can be derived from HSQC and HMBC type spectra. If a file name is specified, the F1 resonance list is redetermined and saved. From HSQC, only protonated resonances can be obtained. An HMBC is often not phase sensitive in F1. So use as F1 resolution no less than the distance between the closest heteroatoms to be resolved. This field is displayed for the HMBC, N15_HMBC, HSQC, COUPLED_HSQC, and N15_HSQC spectrum types. (Key:
[Everything]option is selected, all information determined for every correlation signal is displayed:
All CORRELATION Information =========================== # 6 C 12 C 13 CORRELATION: Fa= 30.038 Fb= 22.013 J= 31.97 Ia1,2 65.8241 555.824 -1.000 65.8241 555.824 67.77 Ib1,2 65.8241 585.838 -1.000 65.8241 585.838 65.30 Fa,Fb 30.0482 30.0382 .2637E-02 22.0183 22.0129 .2362E-0 Pa,Pb -2.21128 -1.98546 .1980 -2.24784 -2.08620 .1836 Ta,Tb .200000 .200000 -1.000 .221400 .221400 -1.000 Ts,J .233319 .233319 -1.000 35.0000 31.9676 .2669 Fd,Pd 52.0665 52.0511 -1.000 -.149412 -.197273 .8358E-0 Tr,Td .100000 .100000 -1.000 .109368E-01 .109368E-01 -1.000 iter 8 Agree .1416 ChiSq .101814574E-01
The information about a correlation labeled
#6 is shown. The first line (
#6 C12 C13 CORRELATION: Fa=30.038 Fb=22.013 J=31.97) is the summary line discussed below. The following lines, from top to bottom, contain the parameters for both spins of the reported correlation:
The six columns of numbers in this table specify, from left to right, the initial estimate, the final value, and the error value for the first parameter, and the initial, final, and error value for the second parameter. A negative error value indicates that the corresponding initial parameter estimate was not optimized during the regression analysis.
EXAMPLE: In the above parameter list, the estimated coupling constant (
J) for this bond in a 2D INADEQUATE spectrum is
35.0000 Hz, and the best-fit determined coupling constant is
31.9676 Hz with a marginal standard deviation (uncertainty) of ±
The last line in the above parameter list gives some statistical information about the reported pattern. Of particular interest is the agreement value (
Agree). It specifies which fraction of the signal power in the experimental correlation area can be explained by the reported pattern. A perfect fit (no noise) would give an agreement value of one. The reported agreement value of
0.1416 (14%) indicates a strong correlation signal given the detection limit of NMRanalyst.
[Summary Lines] option is selected from the
Report About Correlations option menu, the following type of list is generated:
CORRELATION Summary Lines ========================= # 6 C 12 C 13 CORRELATION: Fa= 30.038 Fb= 22.013 J= 31.97 # 16 C 3 C 13 CORRELATION: Fa= 48.792 Fb= 22.016 J= 33.62 # 19 C 2 C 3 CORRELATION: Fa= 54.845 Fb= 48.790 J= 32.66 # 31 C 3 C 4 CORRELATION: Fa= 48.794 Fb= 45.381 J= 29.13 # 32 C 4 C 14 CORRELATION: Fa= 45.383 Fb= 19.644 J= 37.37 # 34 C 4 C 15 CORRELATION: Fa= 45.384 Fb= 19.374 J= 36.93
Each line describes one identified correlation signal and lists the correlation number, the number of each of the correlated 1D resonances,1 both chemical shifts, and for DQF-COSY, HMBC, N15_HMBC, COUPLED_HSQC, and INADEQUATE spectra the coupling constant. For other spectrum types, the average integral instead of the coupling constant is listed.
[Nothing] menu item, neither the summary lines nor the numerical values for detected correlations are reported. (Key:
Report About Correlationsoption menu above.
Non-Correlationstypically are not of interest. So, the
[Nothing]option is selected by default. However, if an expected correlation is not reported, the corresponding summary line can be inspected to see why program
analyzedid not identify this pattern as a correlation:
Non-CORRELATION Summary Lines ============================= # 1 C 7 C 9 No correlation detected: No pattern found. # 2 C 6 C 10 No correlation detected: dI4 Fa # 3 C 8 C 9 No correlation detected: dI4 Fa # 4 C 7 C 8 No correlation detected: dI1 dI4 # 5 C 5 C 11 No correlation detected: dI1 dI4 Fa Fb # 7 C 3 C 14 No correlation detected: dI1 dI4
The best-fit pattern found for fitting area
#1 was not identified as a correlation because at least one of the determined resonance frequencies lies outside the fitting area. The best-fit pattern found in fitting region
#5 was not reported as a correlation because the determined parameter precisions for the A doublet (
dI1) and the B doublet (
dI4) are not sufficiently high and both determined transition frequencies are significantly different from the 1D resonance frequencies (
Fb). For a more detailed description, see the explanation of the
Detection switches in CHAPTER 15: "nD Analysis Workwindow". The determined numerical values for all non-correlations can be displayed by selecting the
[Everything] option. (Key:
The Report workwindow shows a table assigning all multidimensional spectral resonances to the 1D resonances for each spectral direction. For heteronuclear spectra, one table is displayed listing the correlations to the F1 resonances, one listing the correlations to the F2 resonances, etc. For each correlation, the respective chemical shift, coupling constant (or average detected integral), and correlation index number are given:
CORRELATION to RESONANCE 1 RESONANCE 2 RESONANCE 3 ... =============================================================... Shift | Shift J Index | Shift J Index | Shift J Index |... [ppm] | [ppm] [Hz] / | [ppm] [Hz] / | [ppm] [Hz] / |... -------------------------------------------------------------... 56.86| 45.39 31.8 36| 30.05 28.6 52| 9.32 40.9 44|
This 2D INADEQUATE
Connectivity Table fragment specifies that the 1D carbon resonance at
56.86 ppm is correlated to three carbons resonating in the 1D spectrum at
9.32 ppm. The table also shows the corresponding coupling constants (
40.9 Hz) and the correlation index numbers (
#44). The index numbers allow locating further information about the correlations as described for the
Report About Correlations option menu.
Two-dimensional spectra can give rise to ambiguous correlation signal assignments. Ambiguities are reported by the
report program in the
Connectivity Table when the
[Ambiguous Signal Assignment] switch is selected. All ambiguous interpretations are listed with additional messages:
CORRELATION to RESONANCE 1 RESONANCE 2 RESONANCE 3 ... =============================================================... Shift | Shift J Index | Shift J Index | Shift J Index |... [ppm] | [ppm] [Hz] / | [ppm] [Hz] / | [ppm] [Hz] / |... -------------------------------------------------------------... 16.77| 50.48 35.5 105| | | Ambiguity index# 105 and index#(s) 288 16.79| 50.48 35.5 288| | | Ambiguity index# 288 and index#(s) 105
An ambiguity is reported when the same correlation signal is found while examining two overlapping fitting areas. In the 2D INADEQUATE table shown, both ambiguous regions involve the resonance at
50.48 ppm, which is correlated (bonded) to either the carbon resonating at
16.77 ppm (
#105) or that at
16.79 ppm (
#288). The three Hz F1 shift difference between the two resonances (500 MHz spectrometer) is smaller than possible isotope shifts between 1D and 2D carbon resonances, causing both assignments to be indistinguishable. When an ambiguity is encountered, information from other sources is needed to uniquely assign the involved correlations (carbon-carbon bonds). This ambiguity might be a disappointing discovery to the spectroscopist. However, one of the major strengths of NMRanalyst is to reliably detect and report such ambiguities.
[Redetermine 1D Chemical Shifts] switch is mutually exclusive with this
[Ambiguous Signal Assignment] switch, because it removes all but the most probable assignment from a collection of mutually ambiguous assignments. Selecting the
[Ambiguous Signal Assignment] switch displays ambiguous correlations in a different linestyle in the NMRgraph correlation editor. See CHAPTER 19: "NMRgraph: Molecular Correlation Editor" for details. (Key:
[Improper Phase] switch is selected, those correlations with phase values inconsistent2 with the determined phase functions are identified in the
CORRELATION to RESONANCE 1 RESONANCE 2 RESONANCE 3 ... =============================================================... Shift | Shift J Index | Shift J Index | Shift J Index |... [ppm] | [ppm] [Hz] / | [ppm] [Hz] / | [ppm] [Hz] / |... -------------------------------------------------------------... 135.50|132.63 60.7 31|128.54 65.4 12| | Phase mismatch index#(s) 31 12
An individual phase value determined from a 1D spectrum can be shifted by 2 (360 degrees) and two phase values from a single transition determined from a multidimensional spectrum can be shifted by (180 degrees) in two orthogonal directions without changing their appearance. For example, for 2D INADEQUATE spectra, the bond signal model has three phase values (one F1 and two F2 phases). A phase mismatch occurs when one phase is shifted an odd and the other phases an even number of times, or the other way around. The shifted phases and determined phase functions are saved in a plot file as described earlier in the
Output Files section, and the plot can be inspected using the Graphic workwindow.
Even if the
[Correlation Table(s) With Warnings on:] switch is not selected, selecting the
[Improper Phase] switch displays such correlations in a different linestyle in the NMRgraph correlation editor. See CHAPTER 19: "NMRgraph: Molecular Correlation Editor" for details. (Key:
When two correlation patterns identified in different fitting areas are not identical (which would indicate an ambiguity) but both fitting areas partly overlap, the
report program issues an overlap warning if the
[Fitting Area Overlap] switch is selected:
Overlap windows # 105 - # 288 with transitions (A,A) (B,B)
NMRanalyst was designed to distinguish expected spin systems from noise. Overlapping correlation signals can neither be described as isolated signals nor as random noise, and a severe overlap of two or more correlation patterns can possibly lead to a false positive correlation test. The best approach is to acquire the spectrum with sufficient resolution to avoid such problems. If this is not feasible, the overlap warnings allow quickly identifying the spectral regions that could cause misinterpretations of the data.
[Fitting Area Overlap] switch displays such overlapping correlations in a different linestyle in the NMRgraph correlation editor. See CHAPTER 19: "NMRgraph: Molecular Correlation Editor" for details. (Key:
Without selection of this switch, multidimensional resonances are assigned to 1D resonances and the 1D resonance values are shown in the displayed correlation tables and related structure plots. If this switch is selected, new lists of 1D resonances are determined from the multidimensional data. Then the correlation tables and structure plots show resonance frequencies directly determined from the multidimensional spectrum. Selecting this switch removes ambiguous assignments from the correlation tables except for the most likely interpretation. It is recommended to not select this switch if the 1D information was determined from an actual spectrum and to select it if generic 1D information or spectral projections were used for the analysis. For carbon and nitrogen HSQC and HMBC spectra this switch is not shown. Chemical shifts are redetermined if a
Redetermined F1 Resonance List value is specified. (Key:
The panel for the generation of NOE build-up curves is displayed for NOESY and ROESY spectrum types. The shown switch selects the generation of NOE build-up curves. NMRanalyst specializes on the automated analysis of experimental multidimensional NMR spectra. This NOE build-up curve extension provides a simple but powerful way to combine information from a series of NMR spectra. The derived build-up curve slopes are more reliable for the determination of the 3D structure of molecules than the individual signal volumes. (Key:
Mixing Times [ms]fields described above. (Key:
Use this input field to specify a name for the created plot file. If no name is specified,
.plot is used. The two dollar signs in the default name expand to a unique process-ID at run-time. The build-up curve plot is created when the Report workwindow is run with the
[GENERATE NOE BUILD-UP CURVES] switch selected and the
Mixing Time [ms] and
2D Analysis Output File For NOE Spectrum input fields contain valid entries. The numerical analysis results are displayed from the Report workwindow output screen and the created plot can be displayed by the Graphic workwindow. (Key:
NMRanalyst provides the user interface for this build-up curve generation. The
report program calls the
corr2plot shell script for the actual curve generation. Script
corr2plot eliminates curves with negative slope (likely diagonal signals). When three or more mixing time spectra are given, it eliminates those build-up curves containing only one determined volume (possibly a spectral artifact). Each build-up curve shows all determined volumes with error values and the best linear interpolation of these weighted points. The
corr2plot script is a simple text file in Bourne shell syntax. It can be customized as desired.
1The numbers are (if not modified by the user) the position of the 1D resonances in the (decreasing frequency sorted) 1D spectral line list(s).
2NMRanalyst uses an improved version of the algorithm given in Dunkel, R. U.S. Patent 5,218,299 for resolving the 2 ambiguities in 1D and ambiguities in determined multidimensional phase values.