Processing of Routine 1-Dimensional NMR Data. ---Using Topspin ---

Transcription

1 proctopspin.doc >>> 4/11/2012 Processing of Routine 1-Dimensional NMR Data ---Using Topspin --- PART I -- INTRODUCTION 1. Always avoid data processing at a spectrometer unless you are acquiring data at the same time. This provides greater access to the spectrometers and minimizes charges to your account. 2. Log on to the computer. If you are processing data other than at the spectrometer where the data were acquired, first make sure the data are available at the platform you are using. For remote processing transfer the data from the spectrometer computer to PC in X-ray lab a) To process the data at a computer other than the spectrometer computer, you will need to transfer the data from the spectrometer computer. Please see the write-up entitled Transfer of Data for Remote Processing and Archiving for complete directions. b) This write-up in based on using a Windows version of Topspin 3.0. There may be minor differences with different versions. c) Data Processing at the Dell PC in the X-ray lab (Mol. Sci. 1416): 1) Select the picture to the left of the nmr username. Enter the password. 2) Place all NMR data in the appropriate subdirectory below the directory c:\nmrdata. There is a shortcut to the c:\nmrdata directory on the Desktop. The transfer programs can be used to access and create the appropriate subdirectories. or to your own computer b) Data Processing on other computers: 1) The write-up entitled Transfer of Data for Remote Processing and Archiving has details about obtaining the transfer software. 2) You can use any main directory you choose, but the data directory structure must be maintained. c) Data Processing at the spectrometer: 1) This is similar to data processing remotely. The spectrometers all operate under Linux operating systems. This leads to minor differences, primarily for printing. Depending on the age of the hardware, different versions of Topspin are used, which also leads to minor differences. 1

2 2) At the spectrometers, you can change your password. a. On the top menu bar, select Applications Accessories Terminal b. Type passwd. You can then enter a new password. c. The operating system will prevent the use of simple passwords. 3. This is a brief guide that covers only routine NMR processing. Many additional types of data manipulation are available. You are strongly encouraged to consult the on-line help files for further information. A few general comments about the NMR program are given here. a) Start the Topspin program by double-clicking on the Topspin icon. b) Select Help on the top bar to obtain a menu of topics for much more information than is given here. PART II -- PARAMETERS AND FILE HANDLING 1. To start processing you must read in the data and parameters from files that are stored on the disk. The NMR programs use file identification schemes that are subdirectories. It is generally useful to know the directory structure when moving data from one computer to another, e.g. for archiving. a) All data are stored on the disk using the current data set identifiers. Every data set has associated with it a main data directory, user name (USER), data set name (NAME), experiment number (EXPNO) and process number (PROCNO). Identifiers for data sets 1) These identifiers are actually subdirectories. The acquisition parameters and raw data (i.e. the free induction decay (fid)) are located in subdirectories as follows: a. For Topspin 2.1 or older, the data structure must be: /<main data directory>/data/<user>/nmr/<name>/<expno>. b. For Topspin 3.0 or newer, the data structure can be as above, or it can be a simpler form, i.e. /<main data directory>/<user>/<name>/<expno> c. The processing parameters and processed data are located in subdirectories under these called pdata/<procno>. This directory structure is required by Topspin. 2) To locate data using Topspin, the main data directory where your data reside must be in the data browser area at the left side of the main window. If you have your own Topspin, you will have to add the appropriate directory. To do so, position the mouse in this data browser area, right-click, and select Add New Dir. Enter the name of the main data directory. On the Dell PC in the X-ray lab, that will be c:\nmrdata\<spectrometer name>\. Drag desired data set to data area 3) To display a particular data set, expand the main directory by pressing the + on the left of the directory name. Keep expanding until the EXPNO s under the appropriate data set name are displayed. Drag the desired EXPNO into the data area. If more than one process number are present under the selected EXPNO you will be asked to specify 2

3 which one you want. Initially, the data set display will occupy the entire data area. You can reduce the size of this window if you would like to display more than one data set at a time. multiple windows additional process numbers ProcPars tab or type parameter name 4) The data window will initially fill the entire data area. If you place the mouse pointer on a side or corner, then depress and hold the left mouse button you can make the current data window smaller. Dragging a second data set into the blank space in the data area will open a second data window. It will also fill the data area, but you can re-size it such that you can see both windows. If the same nucleus is observed in both spectra (e.g. 1 H), the cursor will track between the two windows even if the data sets are from different spectrometers. Simply click on the window you want to be the active window and the buttons on the menu bars will affect that selected spectrum. Alternatively, select the data window number located in the color-coded buttons at the right edge of the 2 nd row below the title bar. 5) To create a new process number for alternative ways of processing data, type new and specify a new process number (PROCNO). b) Processing parameters can be accessed by selecting the tab labeled ProcPars in the data window. c) Alternatively, if you know the parameter name, you can simply type its name and either change or confirm the value without going through the menu. PART III -- DATA WORK-UP 1. Obtaining a spectrum from the raw data (free induction decay or FID): 1) If the data have previously been Fourier transformed, you can view the time domain data by selecting the tab labeled Fid. A. A few data manipulations may be carried out prior to converting the time domain data (the FID) to the frequency domain (the spectrum). When archiving data, only the time domain data (i.e. raw data) need to be saved because raw data can be easily reprocessed later and you will be able to change parameters that must be applied to the time domain data. window functions 1) Application of window functions - The free induction decay may optionally be multiplied by a variety of mathematical functions, sometimes called window functions. The choice of function and its associated parameters can be used to produce an increase in either the signal-to-noise ratio (S/N) or the resolution, or for apodization, the removal of artifacts that appear as feet at the base of peaks. For routine processing, it is 3

4 suggested that you apply exponential multiplication using the default value for the parameter lb. This will slightly improve the S/N ratio, broaden the lines by lb Hz, and may provide some apodization. Zero-filling 2) Zero-filling - Zero-filling refers to the addition of data points that all have zero values to the end of the FID prior to Fourier transformation to yield sufficient digital resolution in the spectrum. The parameter si determines the extent to which zero-filling will be used in the Fourier transformation. For routine processing, use the default value for si, which includes some zero-filling. B. Fourier transformation - Fourier transformation (FT) converts the time domain data to the frequency domain. Use a combined command for exponential multiplication followed by FT. ef (if phase constants not yet determined) A few comments about spectral manipulations: Type ef. (If you choose not to apply exponential multiplication, just type ft to carry out the Fourier transformation.) Now that frequency domain data have been obtained, a few comments about spectral manipulations follow: 1) Provided that a spectrum is being displayed you can select what items will be displayed with it, e.g. title, parameters, peak labels. With the mouse pointer positioned in the data area, right-click and then select Spectra Display Preferences in the pop-up menu. Select Apply, then Close. 2) "Buttons" for spectrum manipulation, e.g. expansion, horizontal and vertical shifting, etc., are located in the 3 rd row down from the title bar. Positioning the mouse pointer in a "button" will display a short description of its function just below the mouse pointer. The buttons will be described below with the description in bold letters. Always use the left mouse button to select these "buttons". 3) A very useful mode is available if you put the mouse pointer into the area where the spectral data are displayed, then depress and hold the left mouse button while you drag the mouse to include only the spectral region you want to display. 4) The buttons on the 2 nd row down from the title bar are display manipulations that toggle a grid, axis units, etc. 2. Correcting the phase of the spectrum: phase correction A. The phase correction constants required for a particular data set are processing parameters that are stored with files. The values of these 4

5 constants stored in the default parameter files will not be correct since the phasing is a very sensitive function of the precise tuning of the probe and other considerations. Thus, when you first run a particular sample you must determine these constants. If you determined these constants previously, then the appropriate phase correction constants have been stored with the processing parameters. Subsequent spectra on the same sample may be rapidly phased using these same constants. apk or phase subroutine biggest peak or cursor 0 1 1) Initial determination of phase constants - Type apk for an automatic phase correction. Although this can be slow for large data sets, it works reasonably well unless there are broad lines in the spectrum. Alternatively, to determine the phase correction manually, select the Interactive phase correction button in the middle of the 2 nd row below the title bar. A menu bar for the phase sub-routine will be placed at the top of the data area. a) A red line that extends down to the axis will appear on the biggest peak as a marker. This line indicates the pivot point for the first order phase correction. (Alternatively, place the mouse pointer on a peak you would like to use for the pivot point, then right-click and choose Set Pivot Point from the pop-up menu.) b) On the menu bar in the data area, select the "button" labeled 0. Move the mouse vertically while holding down the mouse button to adjust the phase of the peak at the marker. Next, select the "button" labeled 1; move the mouse vertically while holding down the mouse button to adjust the phase of peaks far away from the marker. When you are done with the phasing, select the Terminate phase mode, save phased spectrum to disk button. If you want to exit the phase subroutine without storing and applying the phase correction, use the last button on the right. 2) Phase correction when appropriate constants have been stored with the processing parameters - Type pk to apply the phase correction constants. This is faster and will be appropriate when processing a series of spectra acquired sequentially. It is in fact required in some types of sequential experiments. B. When correct phase constants are available, it is faster to do the exponential multiplication, Fourier transformation, and phase correction with a single combined command. or efp (if phase constants already determined) Replace the command ef given above for Fourier transformation with efp (or fp if you choose not to apply exponential multiplication). 3. Setting a reference: A. NMR chemical shifts are measured and reported relative to the resonance position of a known substance. It is common practice to assign the chemical shift of an internal standard such as TMS or a solvent peak. An external reference may be set by assigning the 5

6 chemical shift of a different known sample, then using as near the same conditions as possible to obtain the spectrum of the sample of interest. calibrate subroutine sr 1) To manually set the reference, first make sure that the axis displayed is in ppm rather than Hz. Then select the Spectrum calibration "button". Place the red line on the peak to be defined. Press the left mouse button, then input the value that you would like to assign to that peak. You may need to expand the spectrum to be able to accurately position the red line. The sub-routine will be automatically exited upon entering the value. 2) When you set the reference, the parameter sr gets set. If sr is very different from 0, (i.e. hundreds of Hz or more), you were probably not locked on the correct solvent peak. 4. Baseline Correction: A. The baseline of an NMR spectrum is almost never flat. In a spectrum with a high signal-to-noise ratio, this may be of no concern and may be ignored. However, vertical expansion of the data usually reveals some distortion. The situation is often worse when peaks occur near the edges of the spectrum. Baseline correction routines are used to deal with the problem; however, sometimes you may want to consider reacquiring the data using different parameters. bas Auto-correct or baseline subroutine 1) Type bas for an automatic baseline correction. Select Auto-correct baseline using polynomial, then select OK. 2) Alternatively, to determine the baseline correction manually, select the Interactive baseline correction button on the 2 nd row below the title bar. A menu bar for the baseline correction subroutine will be placed at the top of the data area. a) A red horizontal line will appear. The goal is to shape this line to match the current baseline of the portion of the spectrum displayed. b) You can choose a polynomial, sine, or exponential function for the correction. The current function will be displayed in a red box on the right side of the data area. The letters A-E designate the coefficients for the current function. With the mouse pointer located on the appropriate letter, hold down the mouse button and move the mouse vertically to adjust the value of the selected coefficient. c) Once the line matches the shape of the current baseline, select the Terminate baseline mode, save regions button. If you want to exit the baseline correction subroutine without applying the correction, use the last button on the right. 5. Integration: 6

7 A. The ratio of nuclei with different chemical shifts, which can be obtained by integrating spectral regions of interest, can be an invaluable tool for making assignments. The accuracy of integration will be approximately +5% if appropriate parameters have been used. Good integrations can only be achieved if adequate digital resolution has been used for the Fourier transformation and if the peak phases have been adjusted properly. Baseline correction are usually required. int or integration subroutine Define new region button toggles between expansions and defining integral regions bias & slope, if needed calibrate selected integral 1) Type int for automatic integration. Select Auto-find regions, integrate and display result, then select OK. This performs an automatic baseline correction then creates an integral file (called intrng). 2) Make sure you have corrected the baseline before integrating. To enter the integration subroutine, select the Interactive integration "button" near the middle of the 2 nd row down from the title bar. a) If the data were acquired with the sample changer automation software, integrals from the automatic integration will be displayed. To delete these and start over, select the Select/Deselect all regions (toggle) button near the middle of the menu bar in the data window. Then select the Delete selected regions button to the right. b) When you enter the integration subroutine, the mouse pointer can still be used to expand regions of the spectrum. Select the Define new region using cursor button. This toggles the function of dragging the mouse to defining integral regions. To switch back to the expansion function, again select the Define new region using cursor button. c) If the integrals are not horizontal at the sides of the peaks, you can make corrections. (If the phase and baseline corrections are good, this should not be necessary.) Select the Interactive bias correction button and hold down the mouse button while vertically moving the mouse to make the portion of the integrals to the left of the peaks horizontal. Select the Interactive slope correction button and hold down the mouse button while vertically moving the mouse to make the portion of the integrals to the right of the peaks horizontal. d) To set the integral value of a particular integral position the mouse pointer in the region defined, click the right mouse button, and select Calibrate Current Integral from the pop-up menu. e) To adjust the sensitivity of the mouse for the bias and slope adjustments, select the Increase mouse sensitivity or Decrease mouse sensitivity "buttons" on the menu bar in the data window. f) When you are done with the integration, select the Return, save regions button. If you want to exit the integration subroutine without saving the integrals you defined, use the last button on the right. B. When processing data, don't forget that accurate integrations are 7

8 strongly dependent on the use of appropriate parameters for data acquisition. If some peaks are saturated to a different degree than others, errors will occur. Different resonances in decoupled spectra may have intensity changes as a result of the decoupling unless steps are taken at the time of data acquisition to avoid these changes. 6. Peak picking: automatic peak picking manual peak picking a) Automatic peak picking be done by typing pps, but it requires that the parameter mi be set correctly first. Manual peak picking is easy to use and is therefore recommended. b) To do the peak picking manually select the Manual peak picking [.pp] button on the 2 nd row below the title bar. 1) The first button on the left of the menu bar in the data window (which is initially green) is used to define regions for peak picking by positioning the mouse pointer in the data window and holding down the left button while drawing a box. The vertical positions of the top and bottom of the box you draw with this tool will determine the minimum and maximum intensities for peak picking independent of previously set values for mi and maxi. If you want to alter the box you drew, select the Modify existing peak picking range button, the 2 nd one on the left on the menu bar. 2) Unlike in the case for integration, the button on the left end for defining the region for peak picking is initially green and can be used to select a region. If you need to use the mouse for expansions, toggle the button used for drawing the box such that it is no longer green. 3) To start over with the peak picking, right click and select Delete all regions. pc 4) All peaks that are to be included must be at least pc times the noise level (where pc is a processing parameter). If there is no peak label on the peak of interest, type pc to see what the current value is. Input a smaller value. You will have to modify the region you have already set for peak labels, or delete it and redraw the box for the region. If the peak is still not labeled, repeat this process until it is labeled. 5) To save the results of manual peak picking, exit the subroutine by selecting the Return, save changes [.sret] button. 6) The Start distance measurement button on the 2 nd row beneath the title bar is a very convenient way to measure peak separations in Hz and ppm. PART IV -- PLOTTING Title a) To edit the title, select the Title tab. Left-click the mouse in area below the menu bar and type in the title. Select the Spectrum tab to return to the spectrum display. b) To plot the data or create a figure for export, type CTRL P or select the Print active window button in the 2 nd row below the title bar. 8

9 simple plotting more versatile plotting suggested layouts edge of plot selecting objects Edit 1D/2D Edit 1) For simple plotting of the data just as displayed, in the pop-up menu, under Options, select Print active window, then OK. Select OK in the Page Setup window. To select a printer other than the default one for the computer where TopSpin is running, select the Properties button, select the appropriate printer, then OK. 2) The Topspin plot editor is a very versatile tool for plotting and for preparing figures in a variety of formats, e.g. jpeg, pdf, etc. To start the plot editor, under Options, select Print with layout start Plot Editor. a. Under Required parameters, if there is no layout listed or if you want to use a different layout, select the button with the down arrow to the right of the LAYOUT = window, select the desired layout, then select Store. The recommended layout names for routine 1 H and X-nucleus spectra are ucla_1d_1h.xwp and ucla_1d_x.xwp, respectively. Under Use plot limits, select from screen / CY to obtain the horizontal and vertical scaling as displayed on the screen. Select OK. If you do not have a layout specified, the spectrum will not be displayed and you can start from scratch. b. The gray dotted line around the edge shows the limits of the space on the page the current printer will actually use. Remember to keep things you want printed inside this line. c. To select the spectrum, use the tool that looks like this: (In the Windows version of Topspin, this is already selected when you enter the plot editor.) Place the mouse pointer somewhere in the spectrum and left click. You will then be able to perform any of the functions on the bar above the data area. The two you will use most are Edit and 1D/2D-Edit. Be sure to have a look at some of the other tools because this is a very versatile plot editor. i.) With Edit, you can input plot limits, select whether axes, integrals, peak labels, etc. will be displayed. Click on the various tabs in Edit to see what else you can do. ii) With 1D/2D-Edit, you can interactively change the scaling of the spectrum and the integrals. By default, the changes will affect both whenever you enter 1D/2D-Edit, but you can turn one off by clicking it at the top of the menu. iii) You can right click on a selected item to obtain a menu of things you can do, including Edit and 1D/2D-Edit. moving & resizing objects adding an expanded region d. While an item is selected, you can move its position by holding down the middle mouse button and dragging it to a new position. Also, it can be resized by positioning the mouse pointer on one the green boxes on its edge or corner and holding down the left mouse button while moving the green box. e. To add an expanded region to the plot, select the spectrum icon on the left (just below the Title icon), then draw a box, which will contain the full spectrum. Next, select the icon on the upper left that contains small green boxes. i) Select the spectrum you just added and use Edit and/or 1D/2D-Edit to make 9

10 it like you want it. adding a different spectrum adding text print export ii) Alternatively, if you would like to display a different spectrum in that box, select it as described above, then select Edit. Select the Data Set tab. Click on the button labeled Select, which will open the Data Set Selector. In that window, click on Edit, then the spectrum you want, then Append, Apply. Back in the Data Set Selector, select the spectrum you just added, then OK, OK. ABC f. To add text to the layout, select Standard, then the abc tool. Left click with the cursor in the plot area, type in what you want, then left click to anchor the box. g. Once you have the plot as you want it, type CTRL P or select the printer icon. h. To convert the spectrum as displayed to a.jpeg,.pdf, or many other file types, select File. In the submenu, select Export, then specify a location for the file, a filename, and the file type. PART V -- FINISHING UP 1. Delete any files that will not be needed for further processing. deletions a) To delete all experiment numbers (Expno's) of a particular data set Name, right click on the data set Name. You will get the message Are you sure to delete all selected datasets? You will have a chance to cancel if needed. b) To delete only some of the experiment numbers under a given data set Name, right click on the experiment number. You will get the message Are you sure to delete the selected EXPNOs including all their PROCNOs from disk? You will have a chance to cancel if needed. c) To delete only some of the process numbers under a given experiment number, right click on the process number. You will get the message Are you sure to delete the selected PROCNOs from disk? You will have a chance to cancel if needed. 2. Exit from the NMR program and log off the computer. exit log off a) Select the x in the upper right of the title bar. You must confirm that you want to exit. b) When processing data at the Dell PC in the X-ray lab, please select start, then Log Off, then Switch User. 10