Basic Procedure 1D NMR Data Collection

Transcription

1 Basic Procedure 1D NMR Data Collection Log into your UNIX account and start XWIN- NMR program At the prompt login:, type your <userid> (e.g. jdw, mmj, mjt) or click on your group icon (in some spectrometers you will see your group icon instead of login prompt). At the prompt password: type your <password>. To open an unix shell terminal, from Toolchest menu click on Desktop and then click on Open Unix Shell. Start XWIN-NMR window by typing xwinnmr in the unix shell (winterm) command prompt. Pre acquisition set-up On the BSMS keyboard: press LOCK ON/OFF button once to turn off the lock. Press SPIN ON-OFF button once to turn off the sample spinning if the spinning light is on. Press LIFT ON-OFF button once to eject the standard sample from magnet bore. Precaution!!! Do not lean on the magnet during sample removal. Handle spinner with kim-wipe to keep it free from finger oils. Remove standard sample tube from spinner and insert your sample tube into the spinner very carefully keeping the spinner on the depth gauge.

2 Measure correct sample tube height in the depth gauge for 5 mm tube. Place sample tube holding spinner into the upper barrel of the magnet. (Make sure there is sufficient air pressure for sample tube and spinner to float and not drop into the magnet, otherwise take the sample tube out and report to NMR Facility Manager). Using BSMS keyboard: press LIFT ON-OFF button once to lower the sample into the magnet. Wait for green down indicator in the BSMS key board to light up as the sample is safely inside the probe. Create a new data set directory for your experiment The FID, pulse sequence, parameters sets, and processed spectra are stored in the form of following data set directory: /disk_unit/data/user_id/nmr/data_set_name/exp_no/pdata/pr oc_no Type edc in the XWIN-NMR command line to create a new dataset directory for your experiment. Precaution: Make sure that the disk_unit (DU) and user_id (USER) are correct for your group!!! Enter your sample code name in the NAME field (maximum 13 characters are allowed and it cannot contain any meta characters (i.e. *, /, &, etc.). Note: UNIX file system is fully case sensitive, so care must be taken while creating new data directory (allowed symbols:., +, -, = and _). Enter experiment number in EXPNO field. For new sample experiment number should be start with 1. Maximum experiment number allowed is 999. Enter processing number in PROCNO field. For new experiment, it should be 1. Maximum processing number allowed for each experiment is 999.

3 Click on SAVE tab and the new file name should appear in the Data set window. Read in appropriate parameter set Type rpar and select desired parameter set from the displayed list and click on copy all tab to copy all parameters for that particular experiment. alternatively, type rpar <parameter_set_name> all e.g. rpar protonstd all Some of the most commonly used parameter sets for routine 1D NMR experiments are as follows: 1. Routine 1 H NMR: protonstd 2. Routine 13 C NMR with 1 H decoupling: carbonstd C DEPT: dept45, dept90, dept135 Locking your sample To lock the field using deuterium signals from your deuterated solvent, lock parameters such as FIELD, LOCK GAIN, LOCK POWER and LOCK PHASE need to be optimized either manually or with software. Type solvent and input name of your deuterated solvent (cdcl3, cd2cl2, c6d6, meoh, dmso, d2o, acetone, etc.) into the pop window and. Type lopo to load solvent specific parameters. alternatively, in DMX and DRX spectrometers, type lopoi and select desired solvent from displayed list Type lopoi solvent_name e.g. lopoi cdcl3 Type lockdisp to open lock display window.

4 Type rsh currshim to load optimized current shim file. In the lock display window (shown in right figure) you should see a peak that rings down as the system sweeps past the resonance frequency in continuous wave mode (one scanning left to right and other scanning right to left). If the start of the signal (decaying ringing pattern) is not in the center of the lock display window, try to align start of the ringing pattern to the center of the lock display window, on-resonance signal as shown in the figure. Special Case: If there is no extended ringing pattern then the shims are really bad and you might find it hard to lock on the signal. If needed, adjust x, y, z, and z2 shims to obtain an extended ringing pattern. Note: If you don t see any ringing pattern, the field/frequency of the lock signal might be outside the displayed window, try adjusting the FIELD value (press FIELD button and turn the knob either clockwise or anti-clockwise) to get the ringing pattern back to the center of the display region. You may need to increase SWEEP AMPLITUDE for this purpose (default value is 2.0). Activate the lock circuit by pressing the LOCK ON/OFF button once on the BSMS keyboard. When the LOCK ON/OFF button light stops flashing the lock circuit is on and you should be see only two lines one scanning from left to right and other from right to left.

5 At this time, it might require to optimize LOCK GAIN, LOCK POWER and LOCK PHASE to obtain reasonable lock level (middle of the lock window) without saturating the signal (line moves in a straight line rather than up and down). Note: Make sure noise level of the lock signal is not very high; otherwise add more deuterated solvent to your sample. If you don t have any deuterated solvent in your sample tube, then to record a spectrum without locking the signal, you need to press SWEEP button once to turn off the field sweep and record spectrum unlocked. In this case you need to shim your sample by FID shimming in gs mode (details in shimming section). Shimming for homogeneity Selection of shim button (e.g. X, Y, XZ, YZ, Z 1, Z 2, Z 3 and Z 4 ) on the BSMS keyboard is slightly different in different spectrometers and you should contact NMR Facility Manager to show you how to use any set shims in a particular spectrometer. Adjust X, Y, XZ, and YZ shims first and optimize the lock level (remember each shim is dependent on other, so when you change one shim other shim changes so you need to practice how to optimize shims). Note: Detailed description for shim optimization procedure is described in a separate section During optimization if lock signal goes off the screen, reduce the lock level by pressing LOCK GAIN button once on the BSMS keyboard and turning the flywheel in counter clockwise direction to bring the lock signal back to middle of the lock window. Click on SPIN ON-OFF button on BSMS keyboard once to start spinning the sample and wait till it stops flashing that indicates that the spinner along with sample tube spins at its desired speed (default value is 20 Hz). Adjust Z 1 and Z 2 shims iteratively to optimize the lock level (Z 1 shim changes most with different samples). Note: Always ensure the FINE button light is on while adjusting Z 1 and Z 2 shims. When finished shimming, press STDBY button once.

6 Tuning the probe Attn: Not recommended for routine operation. Do no perform this operation without the clear instruction from NMR Facility Manager. Type wobb to start tuning/matching operation. Type acqu to change the display in XwinNMR window to acquisition window. Now you should see the wobble display curve (for observe nucleus) as shown in the next page figure. Rotate/adjust rods/sliders under the probe to optimize both tuning and matching (as shown in the figure below). For some of the spectrometers, it might be hard to see the display on the computer monitor while adjusting under the magnet, in that case, look into the HPPR display. During mismatched tuning and matching, HPPR display should show green, yellow and red LED lighting in both horizontal (tuning) and vertical (matching) directions. In case of optimized tuning/matching condition, you should see only 1 or 2 green LED lights in either direction. If you also have to tune decoupler nucleus, then click on wobb-sw tab in the left side menu, a new pop-up window appears with change nucleus option, type y (tune display will automatically change to decoupler nucleus).

7 Optimize decoupler tuning and matching as described above. Once both tuning and matching are optimized, type stop to end the process. Optimization of acquisition parameters Type eda to view all the acquisition parameters and edit. Type ased to view acquisition parameters specific to current pulse sequence and edit. Alternatively, individual parameters can be edited by typing parameter name and editing its values in the pop-up window. Type ii and wait for the message ii: finished to set important changes to the acquisition parameters if any and test all communication before starting data acquisition. Note: If ii failed, there is communication problem in the system, immediately report to NMR Facility Manager and write a note in the Log book. Type expt to estimate total experiment time if performing any long term or 2D experiment. Type rga to adjust receiver gain. This might take few minutes, wait for the rga: finished message. Type zg to start the acquisition. Attn: Make sure there is no mismatch (red light on MIS) sign on top of the BSMS keyboard that displays pulse activity (forward/reflected power), ADC turn-on time, etc. during acquisition (In AMX spectrometer this display is right to your display monitor) [If you see any mismatch, report to NMR Facility Manager]. Type acqu to view FID (free induction decay) and also acquisition status (e.g. number of completed scans and remaining time). Observe data during experiment Type tr to transfer data from spectrometer cpu to host computer disk.

8 Type fp to perform Fourier transformation and phase correction using current values stored in the file for quick viewing of the spectrum quality. FT Type apks to perform automatic phase correction. Check the solvent signal by expanding the solvent signal region (except in case of D2O) or any other known peaks to verify Lorentzian line shape. Note: If the peak shape is not perfectly Lorentzian, you need to optimize shims again depending on the nature of lines (some of the bad line shapes and associated shim are shown in shimming chapter). Type stop to end the acquisition. Start shimming and once you finished shimming, restart the acquisition by typing zg and follow steps after that. If you are satisfied with line shape, let acquisition to finish (wait for checklockshift: finished message). Alternatively, you can halt it at any time by typing halt (if you have enough transients to get reasonable signal-to-noise for your smallest signal). Processing acquired data Type edp to view all the processing parameters and edit parameters if needed. Alternatively, individual parameters can be edited by typing parameter name and editing its values in the pop-up window. Type efp to process data. (efp = em + ft + pk) o em (exponential multiplication) defined by parameter lb default value of lb for 1 H = 0.3 and 13 C = 1.0

9 o o ft (Fourier transformation) pk (phase correction with stored PHC0 and PHC1) Phase correction Click on the phase tab in the left panel of the XWIN-NMR window and a new subroutine appear as shown. before phase correction after PH0 correction after PH1 correction Click on biggest tab once, a dotted line will appear under the biggest peak of the spectrum. Increase the vertical scaling of the spectrum to better visualize phase difference.

10 Hold LMB on PH0 tab: drag the mouse up or down to adjust zero-order phase. Phase the biggest peak (marked with dotted line) with this operation. Hold LMB on PH1 tab: drag the mouse up or down to adjust first-order phase. Try to phase the left most (down-field) peak with this operation. Note: If there is any phase variation repeat two above steps iteratively till spectrum looks properly phased. If it became worse, reset the phase correction routine by clicking once on biggest tab. Once you are satisfied with the phase of the entire spectrum, click on return tab and click on save & return tab in the new pop-up window. Baseline correction Type abs to perform automatic base line correction (i.e. flatten the baseline). Before base line correction After base line correction Note: Use this command if you have a bad base line in your spectrum (such as hump or tilted base line). Alternatively, type basl to perform manual baseline correction. A new sub-menu appears. Hold LMB over A, B, C, D and E tabs and move the mouse up/down to match the shape of the blue line to the distorted spectrum baseline. Click on diff tab to perform baseline correction. After finished, click on return tab and save & return tab in the pop-up menu. Spectrum calibration Select and expand (horizontal expansion) the peak you want to calibrate. Note: To perform horizontal expansion, click LMB once anywhere in the

11 spectrum, a white arrow now will appear tied to spectrum, click MMB once on the left side of the region you want to expand and click MMB once on the right side of the region. Now a spectrum with only expanded region will appear on the screen. In general TMS peak is used to calibrate 1 H, 13 C and 29 Si spectra in which respective signals of TMS appears at 0.0 ppm. However, if there is no TMS, use the solvent peak to calibrate the spectrum. 1 H and 13 C chemical shifts of common deuterated solvents are provided in a chart (look for this chart near your keyboard). Click the calibrate tab in the left panel of the XWIN-NMR window Move the cursor inside the XWIN-NMR window and click MMB on top of the selected peak. A new pop-up window appears with current chemical shift value of the selected peak. Type <new value in ppm>. Click on tab to display the entire spectrum. Integration Automatic integration (software will determine the peak region) can be performed by abs command. However, it is advisable to perform manual integration for more accurate integration. Click on integrate tab in the left panel of the XWIN-NMR window and new sub-routine appears as shown. Click LMB once anywhere in the spectrum region, a white cursor found tied to the spectrum. Click MMB once on the left side (point from which peak starts to grow) of the left most peak (if solvent peak is the left most, then discard it, choose the next peak). Click MMB once to the right side (point where peak merges into the base line) of the right most peak (except TMS)

12 Now an integral will appear over the entire spectrum. Double click LMB anywhere under the integration region to activate the current integral (marked by white cursor on the right edge of the integration curve). Adjust bias (beginning of the integration curve, curvature of the integral) and slope (end of the integration curve, slope of the integral) if integration curve is not parallel to the base line over the noise region (increase vertical scaling of the integral for better view). Note: you can adjust bias and slope by holding LMB on respective tab and dragging it up or down. To break integrals into regions, click LMB once to tie the white cursor to spectrum. Click MMB once on the immediate right of the left most peak/multiplet and once on the left edges of the next peak/multiplet. You will see, now a separate integral is selected for left most peak. Repeat this process till you select all the peaks/multiplets for the entire spectrum (you may have to zoom out and zoom in using these tabs and also use these tabs for moving peaks in horizontal plane). a white cursor appears on top of that integral curve. After all peaks/multiplets are defined by different integral, find out an integral for calibration (an isolated peak with known proton/carbon count) and double click LMB under that peak, Click LMB on calibrate tab in integration sub-menu.

13 A new pop-up window appears with current integral value. Type <new value>. Once you are satisfied with all the integrals, click on return tab and click on save as intrng & return tab in the new pop-up window. To obtain printed list of all integrals, type li. Peak picking Click on utilities tab in the left panel of the XWIN-NMR window and new sub-routine appears as shown. Click LMB on MI tab and slide mouse up/down with no buttons pressed, to move horizontal blue line to top of the smallest peak that you want to have chemical shift labeled (Make sure this line not touching noise level of the baseline at any point). Click LMB to save and return. Click LMB on MAXI tab and slide mouse up/down with no buttons pressed, to move horizontal blue line to top of the largest peak that you want to have chemical shift labeled (this step is optional and required only if you want to discard any large peaks/multiplets from the peak picking list). Click LMB to save and return. Click on return tab. Note: Peaks below MI axis and above MAXI axis will be not be labeled by the peak picking operation. To obtain a printed list of the labeled peaks type pp. Setting title for current data set Type setti and a new editor window appear.

14 Enter title for the current experiment along with necessary details (may be some note/comment regarding experiment). Then select save from File menu followed by exit. Define plot region and plotting Click on dp1 tab (main XWIN-NMR menu), a new pop-up window appears with current values for F1. Type down-field (left) limit (F1) of the plot region <value in ppm>. Type up-field (right) limit (F2) of the plot region <value in ppm>. Answer the question Changes y-scaling on display according to PSCAL? o Type y, if you want to plot the spectrum with same height as displayed on screen. o Type n, if you want to define the height manually with CY parameter. o To adjust CY value, click on utilities tab, click on CY tab, and slide mouse up/down with no buttons pressed, to move horizontal blue line to top of the peak for which you want to set vertical scaling and click LMB. o A new pop-up window appears, type <new value CY value in cm>. o Click on return tab. o XWIN-NMR window next shows newly defined plot region. Type view to preview the plot layout. After preview, click quit tab to exit from plot layout window. Type plot to obtain a printout. Click on seen tab in new pop-up window. If you want to plot expanded region, redefine plot region as described above and plot again. Logout procedure Press SPIN ON-OFF button to stop sample spinning. Press LOCK ON/OFF button to cancel lock hold.

15 Press LIFT ON-OFF button once to eject your sample (carefully remove sample from magnet bore). Replace your sample with standard sample (0.1% ethyl benzene in CDCl3). Place standard sample holding spinner into the upper barrel of the magnet. Press LIFT ON-OFF once to insert the standard sample into the magnet. Load lock parameter for CDCl3 by typing lopoi cdcl3. Adjust the FIELD to bring the start of the ringing pattern to middle of the lock display window. Press LOCK ON-OFF button to lock the signal. Type rsh currshim to load standard shim file. Type edc, create a new data set with name test (This will ensure that your data set is not over-written by the next user in your group who uses the spectrometer). Type ii to reset all changes to default values (make sure test 1 1 data set is set for protonstd experiment). Click on quit tab inside the lock display window. Select exit from File menu of XWIN-NMR window. Click OK in the pop-up window. Click on Desktop from Toolchest and click on Log Out. Click Yes in the pop-up window. Fill-in log book with all details (write down if you find any unusual activity).

16 Commonly used Keyboard commands Most of these commands are described above. This is a compilation of all commands (including their abbreviation) that can be typed from your keyboard. Parameter setup: edc eda ased edp wrpa re edit current dataset to create new data set or load existing data set edit nuclei and spectrometer routing edit acquisition parameter used in current pulse program edit processing parameters copy current dataset specify new experiment number to copy read new data set specify new experiment number Data acquisition: lockdisp lopoi rpar rsh wsh gradshim ii acqu wobb rga zg ns ds d1 sw o1p o2p td expt go tr halt stop gs edte display lock window select parameters for specified lock solvent read standard/user defined parameter set read stored shim file write current shims to a file (you need to provide a file name) display gradient shimming window initialize interfaces (verifies all communication) display acquisition window (shows FID during acquisition) start wobble routine for probe tuning receiver gain adjustment (set receiver gain automatically) zero fill current data set and start acquisition number of scans number of dummy scans recycle delay size of spectral window in ppm transmitter offset in ppm for observe nuclei transmitter offset in ppm for decoupler nulei time domain data points display length of the experiment start data acquisition (it will add to existing data) transfer acquired data to disc for processing after next scan halt data acquisition after next scan stop data acquisition immediately Real time observation of the FID in the acquisition window with option of interactively manipulating offset, delay, pulse widths, power levels, etc. (Note: it doesn t accumulate any data) display temperature controller window

17 Data processing and plotting: setti ft em lb gm pk ef fp efp basl pscal cy mi maxi pps pp view plot dual set title of the current dataset Fourier transformation exponential multiplication controls amounts of exponential multiplication gaussian multiplication controlled by parameter gb and lb phase correction using stored PHCO and PHC1 values exponential multiplication and Fourier transformation Fourier transformation and phase correction using stored PHCO and PHC1 values exponential multiplication, Fourier transformation and phase correction using stored PHCO and PHC1 values enter baseline correction submenu define vertical scaling of the spectrum for plotting height of the peak defined by cm minimum threshold for peak picking maximum threshold for peak picking peak picking with output on screen peak picking with output on paper view plot output on screen plot spectrum on paper enter dual display window need to provide name, expno2 and procno2. 2D Data acquisition and processing xfb xf2 xf1 abs2 abs1 2D Fourier transformation in both dimension Fourier transformation of 2D data in F2 dimension only Fourier transformation of 2D data in F1 dimension Base line correction in F2 dimension Base line correction in F1 dimension Common unix commands lpstat -t ps -ef shmrm pwd cd <dir name> uxproc provide plot status Lists active processes kills all hanging XWIN-NMR processes show current directory Change directory Check for NMR processes

18 Location of common files and directories pulse programs gradient programs shaped pulses cpd programs frequency lists parameter sets shim files user data /u/exp/stan/nmr/lists/pp /u/exp/stan/nmr/lists/gp /u/exp/stan/nmr/lists/wave /u/exp/stan/nmr/lists/cpd /u/exp/stan/nmr/lists/f1 /u/exp/stan/nmr/lists/par /u/exp/stan/nmr/lists/bsms /<disk unit>/data/<user id>/nmr/