核磁共振氫譜解析

1、Using Mnova to Process, Analyze and Report NMR and LC/GC/MS on Your Desktop Chen Peng, PhDDirector of Business Development, US & ChinaMestrelab Research SLSan Diego, CA(858) 736-4563chen.Version 6.2March 20111Use Mnova NMR toOpen and transform your NMR dataProcess, analyze and report a 1H spectrumUs

2、e Mnova NMRPredict Desktop to Predict 1H and 13C and verify your structureAssist you assign peaksUse Mnova MS to Open your LC/MS raw data and browse MS and UVVerify your proposed structuresContents* Only the most routinely used features are covered. More advanced features, as well as Mnova DB (datab

3、ase), ASV (auto structure verification) and Assign plugins are not covered in this tutorial.NMRNMRPredictMSDBASVAssignMnova: An integrated system for analytical chemistryMnova is compatible with Mac, Windows and Linux2Mnova NMRQuickly process and analyze 1D NMR, and report your chemical shifts and J

4、-couplings in journal formatProcess, analyze and assign multiple 2D spectra together with 1D*Advanced tools for automation, quantitation, reaction monitoring, diffusion & relaxation studies*Mnova NMR license required*Those features are not illustrated in this tutorial. See Mnova Help Contents for mo

5、re detailsNMRNMRPredictMSDBASVAssignMnova: An integrated system for analytical chemistry3To open and transform your NMR dataChoose File | Open to open the fid the raw dataOr drag an fid Windows Explorer to Mnova *Mnova automatically transforms the raw frequency domain(including Windowing function, F

6、ourier transform, phase correction etc) *You can drag multiple folders that contain fid (or ser) to Mnova to open multiple spectra simultaneously. *Parameters from the raw data are used for processing. You can view or change the processing parameters by choosing Processing | Processing Parameters. S

7、ee Help Contents Processing Basics for more detailsDrag & drop4Click for phase correction if peaks are not symmetric*Click for baseline correction if baseline is not zero *Click to calibrate the chemical shift reference if the solvent or TMS peak is not at the right ppm *Click the arrow next to the

8、tool icon for options. See Help Contents Processing Basics for more detailsTo correct phase, baseline & reference5Zoom in/Zoom out (or press Z) *Zoom outFull spectrum (or press F)Manual Zoom in to defined ppm rangePan spectrum (or press P)*Expansion click&drag to draw an inset (or press E)Fit to Hei

9、ght (or press H)Increase Intensity (or rotate mouse wheel)Decrease Intensity (or rotate mouse wheel)Crosshair Cursor (or press C) for measuring J-couplingsCut (or press X) to hide parts of the spectrumTo visualize your spectrum*Press Z several times to toggle between horizontal/vertical/box zoom* Pr

10、ess P several times to toggle between free/horizontal/vertical panningPress E, then Click and drag to define the range for the inset6To analyze and report multiplets in H-1 NMRMnova provides several approaches to multiplet analysisManual: click-and-drag to pick each multiplet interactivelySemi-auto:

11、 pick peaks and integrate first, then do auto multiplet analysisFully automatic multiplet analysisIn either case you can refine the results interactively, and report them in selected journal or patent formats7To analyze multiplets manuallyPress Z and zoom into one or more multipletsPress J to switch

12、 to Manual Multiplet modeClick and drag to include peaks for a multiplet*Double click on the multiplet label to open the Multiplet Manager Panel (see next slide)Choose View | Full View to show the Full View window if you want (see next slide)Click and drag to define the range and peak picking thresh

13、old for the doublet* Mnova 6.1 or older: You can only define the range but not the threshold. The automatic peak picking threshold is used for picking peaks.8Full view and zoom-in view for multiplet analysisFull View: The whole spectrum and zoom-in area. Drag the blue box to move to other multiplets

14、. (Choose View | Full View to open it)Manual multiplet analysis: Press J, then click and drag to define the range and peak picking threshold for a multiplet. Multiplet Manager shows the properties of the current multiplet picked. (Double click on a multiplet label to open it)Multiplet label: Hover t

15、he cursor on it to see peaks. Use the bar to split a multiplet 9To refine multiplet analysis results Hover the cursor on the multiplet label to show the peaks in the multipletClick/drag the small red box to split the multiplet at desired placeYou can also click/drag the small green boxes to change t

16、he range of a multipletUse the small red box to split a multipletUse the small green boxes to change the range of a multiplet10To refine multiplet analysis resultsSelect the Add Multiplet Peak toolClick SHIFT key once to switch to free peak picking modeClick on the shoulder peak at around 3.09 ppmCl

17、ick here to pick the missing shoulder peak. Note: you may need to click SHIFT key to exit the auto peak picking mode to pick it. 11To refine multiplet analysis resultsThe new peak is added to multiplet C because it falls in the range of multiplet CClick on that peak and drag it to multiplet BIt belo

18、ngs to multiplet B now, and the multiplet patterns are auto. updatedDrag this peak to the label of multiplet B12Multiplet ManagerUse the Multiplet Manager to inspect and change the properties of a multipletMove to the Previous/Next multipletDelete the current multipletAdd/Delete multiplet peaks Prop

19、erties of the current multipletNormalized integral and nuclide countsChemical shift range of the multipletThe current # of protons included in the multiplets. 13To report multipletsClick Multiplet Analysis | Report to report the results in a journal format:To change journal format: choose View | Tab

20、les | Multiplets to display the Multiplets Table. Click Setup Report1H NMR (400 MHz, CDCl3) 8.62 (d, J = 4.5 Hz, 1H), 7.95 (d, J = 9.2 Hz, 1H), 7.46 (d, J = 4.5 Hz, 1H), 7.30 (dd, J = 9.2, 2.7 Hz, 1H), 7.21 (d, J = 2.7 Hz, 1H), 5.80 5.66 (m, 1H), 5.48 (d, J = 4.1 Hz, 1H), 4.92 (ddt, J = 13.3, 10.3,

21、1.4 Hz, 2H), 3.87 (s, 3H), 3.46 3.27 (m, 2H), 3.18 3.00 (m, 2H), 2.63 (ddd, J = 12.6, 7.1, 3.9 Hz, 2H), 2.32 2.08 (m, 1H), 1.90 (s, 2H), 1.83 1.63 (m, 3H), 1.51 (dddd, J = 12.4, 7.3, 5.7, 2.5 Hz, 2H).Tip: From the Multiplet Table, click Copy Multiplets and then paste the texts to your document. Clic

22、k Copy Table and then paste the spreadsheet to your document. The table can be customized using Setup Table.14Semi-automatic multiplets analysis1st: Do peak picking. Clean up the peaks2nd: Do integration. Clean up the integrals3rd: Do auto. multiplet analysis for the whole spectrum based on those pe

23、aks and integralsFinally edit and report the multiplet results as described in the previous slides15Region and threshold to pick peaksTo pick peaksClick to do auto peak picking. If results are not good, click Options to change the threshold:*Or choose Manual Threshold (or press K), click&drag to def

24、ine the region and threshold to pick peaksChoose Peak by Peak (or press Ctrl+K) to pick one peak at a time* Threshold is the (auto estimated) Noise Level multiplied by the Noise Factor (user-defined). * By default, Mnova automatically locates the peak tops. Click Shift key to turn it off when pickin

25、g shoulder peaks. Tip: Choose Edit | Properties to change the way to display peaksThreshold for picking positive and negative peaks16To integrate peaksClick to do auto integration Double click on an integral curve to popup Integral Manager:Type a Normalized value to normalize the integralsBrowse, de

26、lete, change, split integrals interactively if neededClick and drag the left green box to change the range of the integralTip: You can click and drag an integral curve to move them up or down, and change their sizes. See Help Contents Analysis tools Integration for more details. 17Automatic multiple

27、t analysisClick to do peak picking, integration and multiplet analysis fully automaticallyNext browse and refine the results as described in the previous slidesTips: A suitable peak picking threshold is critical to the performance. See previous slides about it. Peaks and integrals are not labeled. P

28、eaks are shows when you hover the cursor on a multiplet label.By default, the J coupling constants are not displayed in the labels. Right click and select Properties to turn it on. 18Choose Scripts | R to report in a predefined formatClick to generate PDF, or copy/paste to your documentsTo report us

29、ing the R script* You need to install R script. Write to to request for it.Tip: You can copy a molecule from ChemDraw, Isis/Draw or ChemSketch, or open .mol or .sdf files. 19To annotate and report manuallyClick the Annotation Options button at the bottom-left corner of Mnova windowOr press T to inse

30、rt a text boxAll objects can be customized by right clicking on it and then selecting the Properties commandTables of Peaks, Integrals, Parameters etc can be opened by View | Tables. Report from there Tips: *Copy a molecule from ChemDraw or Isis/Draw, or open .mol or .sdf files*Use View | Layout Tem

31、plates menu to generate and apply layout templates, or request an auto formatting script from Mestrelab. *Copy/paste any object(s) to your document with high resolution*Click to export PDF20Mnova NMRPredict DesktopPredict 1H, 13C, 15N, 17O, 19F, 29Si, and 31P spectra Predict and verify a structure a

32、nd do peak assignment interactivelyMnova NMRPredict Desktop license requiredNMRNMRPredictMSDBASVAssignMnova: An integrated system for analytical chemistry21To predict NMR from a structureOpen a new document (File | New) or a new page (Edit | Create New Page)Copy a structure from ChemDraw, Isis/Draw

33、or ChemSketch, and paste to Mnova, or open a .mol or a .sdf fileChoose an option from the Predict menuTips: 1. Choose Molecules | Prediction Options to change settings2. You can turn on/off the atom numbers by right-clicking on the structure and choose Properties. 22To predict NMR & verify your stru

34、ctureOpen your 1H (or 13C) spectrum in a new pageCopy your structure from ChemDraw or Isis/DrawChoose Analysis | Predict & Compare. The predicted spectrum is stacked with the experimental one for visual comparisonHover your cursor on the atom to highlight its predicted peak23To assign NMR multiplets

35、 to atoms (1)Do Multiplet Analysis to get the multiplet labelsDo Predict and CompareChange the stacking mode to “Active Spectrum”, press Shift + Up Arrow Key to make sure the experimental spectrum is displayed (so that the multiplet labels are visible)Hover the cursor on an atom to see its predicted

36、 peak (in blue). Press A, and click on an atom to assignClick on the multiplet label to assign to that atomTips: 1. The predicted spectrum helps you assign peaks, but you dont have to have it for assignment. 2. After Predict and Compare, the two spectra are stacked. In the Stacked Mode, the multiple

37、t labels are not displayed. You have to change to Active Spectrum mode to see the multiplet labels.2. Press ESC to exit assignment mode.3. Choose View | Tables | Assignment to report the assignments. 4. Multiple 1D and 2D spectra can be assigned simultaneously in this way.24To assign NMR multiplets

38、to atoms (2)Hover the cursor on the atom (#8) to assign. The predicted peak is displayed as the blue doublet. Click on atom #8 and then on this multiplet label to assign it to atom #8.25To assign NMR peaks to atoms without multiplet analysisDo Predict and Compare firstPress A, and click on an atom t

39、o assignClick and drag on the experimental spectrum to include the multiplet to assignOr click on a peak top to assignTips: 1. The predicted spectrum helps you assign peaks, but you dont have to have it. 2. Press ESC to exit assignment mode.3. Choose View | Tables | Assignment to report the assignme

40、nts. 4. Multiple 1D and 2D spectra can be assigned simultaneously in the same document.5. Use Predict | Update 1H (or 13C) User DB to save your assignment for improving the prediction (Mnova 6.2 only)Click on the atom (#8) to assign. It highlights its predicted peak. Click and drag to define the pea

41、k to be assigned to this atom (#8). 26Mnova MSMnova: An integrated system for analytical chemistryVisualize your LC/GC-MS data and UV components from various vendorsIntegrate peaks automatically or manually with easy reportingVerify proposed structures by matching mol ion and isotope peaksEnumerate

42、possible elemental compositions from a selected ion peakMnova MS license requiredNMRNMRPredictMSDBASVAssign27Agilent ChemStationMassHunterIon TrapMnova MS: Open raw data automaticallyThermoXcaliburWatersMassLynxBrukerXmassCompassRaw dataDrag & dropABI/SCIEXAnalystJEOLMSQ1000ShimadzuSee for more deta

43、ils and limitations. NMR spectra can be opened in the same document. Molecular structures can be opened as .mol or .sdf files, or be copied from ChemDraw, Isis/Draw and ChemSketch. 28To open your LC/MS dataChoose File | Page Setup | Orientation and change the page orientation to portrait, if you pre

44、fer.Choose File | Open to open any the folder containing the raw data, or drag/drop the folder from Windows Explorer to MnovaMnova automatically converts your data and does peak integration. Drag & dropTICMS trace29To browse the MS tracesClick to switch to crosshair cursor, and click on the TIC to d

45、isplay the MS trace at that retention time. Click to change to appending mode if you want to display multiple MS tracesChoose the Spectrum Selection Mode options to display co-added MS traces:30To browse the UV tracesClick to show the MS Browser PanelChoose the Total UV Absorbance under Traces, and

46、Click to display the UV TICRepeat the above step to display the other UV components if any31To edit and report peak integration resultsPeaks are automatically integrated when you open a chromatogramUse the Peak Detection tool menu to re-detect peaks, add, delete or clear peaks Hover your cursor on t

47、he wedges, click and drag the green boxes to change the range of a peakOr press Shift, click and drag the green boxes to change the baseline of a peakChoose View | Tables | Mass Peaks to display or report the Mass Peaks Table TICSHIFT+Drag32To display extracted ion chromatogram (EIC) from an m/z val

48、ueClick (or choose Mass Analysis | New Mass Chromatogram | Manually)In the New Chromatogram dialog, enter the m/z value that you are interested in, and a suitable TolerancePress OK to display the EICTICEIC at 195.1 +/- 0.25 Da33To display extracted ion chromatogram (EIC)for an MS peakFirst display t

49、he MS trace and zoom into the molecular ion peak that you are interested. Next select Click (or choose Mass Analysis | New Mass Chromatogram | Graphically), click-and-drag around the peak to define a mass rangeAn EIC will be displayed within the mass rangeTICMS at 0.63 min EIC at 194.8-195.6 Da34To

50、confirm proposed structures using Molecule Match (1)Throw in one or several structures by copy/pasting from ChemDraw, Isis/Draw or ChemSketch, or by opening .mol or .sdf files.Click (or choose Mass Analysis | Molecule Match | Calculate).In the Molecule Match Table, click on a molecule to see its mat

51、ching resultsTICMatched Isotope Cluster Chromat.Matched Isotope ClusterMol Match Results35To confirm proposed structures using Molecule Match (2)You can choose Mass Analysis | Molecule Match | Settings to change the settings for Molecule Match.The default settings are for low-resolution MS. Change T

52、olerance to 5-10 ppm if you are using high-resolution MS. Edit the Adducts or Losses if you want.Click to run the Molecule Match again36To confirm proposed molecular formula using Molecule MatchIf you dont have a structure but only a MF, choose the Calculate From Molecular Formula toolEnter one or m

53、ore molecular formulasThe results are displayed in Molecule Match Table37To calculate elemental compositionZoom into the molecular ion peak of a MS traceClick or choose Mass Analysis | Elemental Composition | Calculate. Click on the molecule ion peak. An Elemental Composition Table is displayedClick on a row to see the match of observed and predicted isotope peaksChoose Mass A