NMR FACILITY DEPARTMENT OF CHEMISTRY University of Washington XWINNMR 3.5 TRAINING NOTES. 08/8/2005 rajan paranji

Transcription

1 NMR FACILITY DEPARTMENT OF CHEMISTRY University of Washington XWINNMR 3.5 TRAINING NOTES 08/8/2005 rajan paranji

2 CONTENTS Views of XWINNMR INTERFACE 4 Operating the Bruker Spectrometer FLOW CHART 5 LOGON 6 SAMPLE HANDLING 7 DATA SET DEFINITION 8 PARAMETER EDITING 10 LOCKING AND SHIMMING 12 ACQUISITION 14 PROCESSING FT 15 PHASE CORECTION 16 INTEGRATION 17 PEAK PICKING 19 PLOTTING 21 LOGOUT 26 DATA ARCHIVAL 27 APPENDIX A LIST OF COMMONLY USED XWINNMR PARAMETERS 29 B COMMONLY USED COMMANDS IN XWINNMR 33 2

3 TYPOGRAPHIC CONVENTIONS USED IN THIS NOTES: boxed text corresponds to a button to be clicked with mouse on screen OR a key to be pressed on the BSMS keyboard. LIFT ON/OFF Commands to be typed appear with the following font: edc Mouse buttons are depicted like this : LEFT 3

4 ACQUISITION WINDOW, SPECTRUM WINDOW 4

5 OPERATING THE BRUKER SPECTROMETER LOGON Username Password PLOTTING / OUTPUT xwinplot SAMPLE INSERTION Positioning sample in spinner Inserting (or) ejecting sample LOGOUT Stop spinning Eject sample Logout of XWINNMR DATA SET DEFINITION Creation of new dataset Loading standard parameters DATA ARCHIVAL / BACKUP FTP data to your lab PARAMETER OPTIMIZATION & PREPARATION Tuning and Matching probehead Locking and Shimming Spectral width, FID size ACQUISITIION Receiver Gain optimization Beginning Acquisition Halting/Stopping Acquisition PROCESSING Spectrum size Window function FT and phase correction Integration Peak Picking 5

6 LOGON Username: genuser Password: nmr301 Password for any instrument is nmr followed by the 1H frequency of that instrument. Instrument Name Host Name Used for FTP Base Frequency AV300 av300.chem.washington.edu MHz AV301 av301.chem.washington.edu MHz AV500 av500.chem.washington.edu MHz DRX499 drx499.chem.washington.edu MHz DMX750 * dmx750.chem.washington.ed u MHz * For DMX750 alone, different research groups are provided with individual login accounts. Contact the Facility Manager for details. 6

7 SAMPLE INSERTION To Lift the sample out of the magnet: Press the LIFT ON/OFF key on the BSMS keyboard OR type ej on the command line and enter To insert the sample into the magnet again: Press the LIFT ON/OFF key on the BSMS keyboard again OR Type ij on the command line and enter. Points to Note: When the sample is seated properly inside the magnet, a GREEN colored LED next to the label DOWN will be lit on the BSMS keyboard. When the sample is floating on top of the magnet, a GREEN colored LED next to eh label UP will be lit on the BSMS keyboard. While the sample is being transported in between DOWN and UP positions, a RED colored LED next to the label MISSING will be lit. This light should not be lit at any other time. Even after injecting the sample into the magnet, if you see this RED LED on, there is some problem with the sample transport system. PLEASE CONTACT THE FACILITY MANAGER OR ASSISTANT IMMEIDATELY. 7

8 DATA SET DEFINITION Enter the command edc. Next to user, type your name Next to name, enter a meaningful name for the dataset. Next to expno, enter a numerical value (1, 2,...10,...) Next to procno enter a numerical value (1, 2,...10,...) Click on SAVE 8

9 DATA STRUCTURE OGRANIZATION IN XWINNMR $NMR /opt/xwinnmr exp conf data prog bin User directories User 1 User 2 User 3 nmr Dataset 1 Dataset 2 Dataset n Experiment Number pdata Processed data number

10 PARAMETER OPTIMIZATION & PREPARATION Assuming that you are going to record a 1 H ( i.e. proton) spectrum first, proceed as below: Type proton on the command line Type ased to access the window that shows you the parameters that are relevant to a 1D proton experiment. Modify the parameters that are of importance to you. Click the SAVE button to exit the table. Enter edte on the command line. Choose the appropriate temperature. Wait for the temperature to 10

11 stabilize. You must wait at least 5 minutes. Enter wobb on the command line. Look at the wobble curve. The cusp of the wobble curve should touch the bottom and it should also be aligned with the blue vertical line at the center of the screen. edte WINDOW wobb CURVE 11

12 Locking and Shimming Enter lock Choose your solvent from the drop down list. Wait for the Automatic Locking process to complete. You will see the message Lock finished just below the command line. Enter lockdisp as shown here. LOCK DISPLAY SOLVENT LIST Start shimming. You can use the BSMS keyboard to vary the different shim currents. If you prefer not to use the BSMS keyboard for this, there is another option. Enter the command bsmsdisp on the command line and you will be given a graphical interface that will have buttons that mimic all the functions of the BSMS keyboard. 12

13 BSMS DISPLAY WINDOWS: 13

14 ACQUISITION Enter rga For proton observation the receiver gain i.e. rg can be anywhere from 10 to 250. If the receiver gain is a larger number like 1800, for example, this indicates that the signal is weak. This implies a problem somewhere. Enter zg o You can switch to the acquisition window by typing acqu on the command line. Here you will be shown a status window while the data is being acquired. Status window shows how many scans are complete and how much more time is left to complete the data collection. o Wait for the message Checklockshift finished to appear below the command line. o You can enter at any time during the acquisition, the command tr to transfer the FID that has been recorded so far and process the same to see how your experiment is progressing. This is useful, for instance, in 13C experiments. 14

15 PROCESSING 1. FT Enter efp to process the FID and get a spectrum. efp actually stands for three operations : em + ft + pk em multiply the FID with an exponentially decaying function. ft perform fourier transformatioin of FID pk apply phase correction as defined by the parameters phc0 and phc1 What is shown above is a default command which serves well to get solution NMR of small molecules. But you can change these behaviors if you need to. You can choose a different window function by invoking the command winfunc on the command line. 15

16 2. PHASE CORRECTION Click on the phase button to enter the phase correction routine. Click on the cursor button. The pointer will turn into an inverted white colored arrow that is tied to the spectrum. Move it to the rightmost part of the spectrum. Use the PH0 button to interactively adjust the phase till all the peaks look symmetric and positive (Lorentzian lineshape). If necessary, use the PH1 button also. Click on return button. You will get three buttons of choice. Choose Save & Return. 16

17 3. INTEGRATION A: Basic Steps Click on the integrate button to enter the integrate routine Click on LEFT mouse button anywhere inside the spectrum. The pointer will turn into an inverted semi-transparent arrow that is tied to the spectrum. To integrate an isolated peak, move this cursor onto the left side of the peak. Click on the MIDDLE button. The inverted arrow now leaves a copy of itself, in the form of an opaque white pointer that is fixed at the position where you clicked. Move the transparent arrow to the right side of the peak. Click the MIDDLE mouse button once again. Now, you will see a blue colored integral appear, between the limits you have selected. You simply have to repeat this operation for integrating all the peaks. Once you are done, click the LEFT mouse button to come out of this integrating mode. 17

18 B: Calibrating integrals: Double click under the integral you want to choose as your standard peak for calibration (example: a methyl group peak corresponding to 3 protons). A white opaque arrow sticks onto the integral you have chosen. This is called the current integral. Click on the calibrate button. You will be given a dialog box. You can enter the number of protons that gives this integral. In the example above, you will enter 3. All the areas shown for other integrals will be recalculated based on the above calibration. C: Saving and Recalling integrals: Once you are finished integration and calibration, click on return button. Among the choices given, you must choose save & return now. Then only the integrals are stored with your dataset. Let us say you have already integrated the spectrum a few days ago. How do you recall those integrals now and see on the screen? o After you click the integrate button in your main spectrum window, you can click on the pull down menu File. o You will see the first choice in this pull down menu as read intrng. If you click on this, you will get your saved integrals displayed once again on the spectrum. D: Hard Copy output of Integrals : You can print the integrals as a list. Enter the command li on the command line. By default the list of integrals goes to the currently chosen printer or plotter. E: Comparing Integrals Across spectra : Instead of choosing a given peak under the current spectrum for calibration, if you click on lastcal, all the integrals will be calibrated in reference to the previous integration operation. This can be very useful in kinetics studies or comparing two different compounds as they evolve over time. 18

19 4. PEAK PICKING A: Basic Steps Enter pscal global See if the vertical scale of the spectrum is in units of cm. If not, click on the YU button. This should switch the vertical scale units to cm. Click on the utilities button. You will be taken into a display shown below. Click on MI button. A horizontal line appears. If you click and hold the mouse cursor on the position of MI button and move up and down, the horizontal line also moves. Position this line where you want the lower threshold to be. Peaks that appear below this line will not be considered for picking. Now click on MAXI button. Another line appears and this you can use to define the higher threshold. Peaks that are taller than this level will not be considered for picking. Now, enter pps to see a listing of the picked peaks in a separate window. You can send this list to the printer if you choose. 19

20 B: Optimizing Peak Picking Enter pc. By default pc is 1. If you want more number of peaks to be considered for picking between MI and MAXI, decrease the value of pc below 1. If you want lesser number of peaks to be picked, increase the value of pc to something larger than 1. 20

21 PLOTTING XWINPLOT EDITOR XWINPLOT is a stand alone Plot editor that is quite versatile and user friendly. It is called a WYSIWYG plotter. What You See Is What You Get. Any existing XWINNMR dataset can be imported into XWINPLOT and edited. When you start XWINPLOT from within XWINNMR, the current dataset is automatically loaded. A: Basic Steps: Enter xwp from XWINNMR. The current spectrum is loaded and XWINPLOT window opens. An example is shown below. In the simplest case, once you have opened the XWINPLOT with the current data set, you just have to click on the File pull down menu, choose print and your spectrum will be plotted on the plotter. That s it! 21

22 B: Optimizing XWINPLOT Features: XWINPLOT BUTTONS AND THEIR FUNCTIONS: Select a Graphics Object (Spectrum, Title Box, etc.) Expand a region of the spectrum using mouse Zoom onto a region of the display On Screen only Insert Title from the current data set Insert the list of parameters for the current data Single spectrum display mode Spectral Array display mode Text insertion box Any of the above buttons can be clicked once to choose that particular mode of editing. The mode you have chosen currently, will be displayed in the lower left corner of the xwinplot area, as shown below: To switch between modes, you have to click the corresponding button once. 22

23 XWINPLOT shows a white area in a cyan background. This is a virtual sheet of paper on which you will place your graphics objects. By default, XWINPLOT includes the following objects of display, when you open it with a valid dataset. Spectrum in landscape orientation List of parameters used in a two column format on the right side of the spectrum Integrals Title of the spectrum from the dataset. The default behavior can be changed completely by editing the objects of display individually. Do the following for the editing. 1. Click on the button. Move the cursor onto the spectrum display and click once. The spectrum area is surrounded by green handles or hot spots, like below. 2. Now click on the button on the top right corner, next to the 23

24 button. You will get an edit window like below: Size of the spectrum window displayed. Edit font type, sizes, curve thickness, etc. Size and position of displayed integrals X-axis Units 1. If you want peaks to be displayed, you can turn on the Show Peaks button. 2. If you want to change the limits of X-axis, you can modify the Xmin, Xmax range. 3. For Y-axis limits, edit the Ymin, Ymax entry. 24

25 4. Once you are finished, click OK or Apply You can edit pretty much any other graphics object that was placed inside the white area of XWINPLOT by using the edit mode. 25

26 To end your run do the following : LOGOUT If you are using a temperature that is different from ambient temperature, then set the temperature to 298 K. Stop spinning the sample. Click on the LOCK button the BSMS keyboard to end locking. Click on the LIFT ON/OFF button to eject your sample. Transfer the dummy sample into the spinner and insert that into the magnet. Enter exit on the command line. Click OK to the questions asked. XWINNMR automatically logs you out of the system once the session is over. You must do this to enable the next user in line to use the instrument. If you do not log out there is a very real possibility of your data being overwritten by the next user, unknowingly. 26

27 DATA ARCHIVAL & RETRIEVAL XWINNMR stores all the FID that are collected but automatically erases the spectra once they are a week old. To regenerate the spectrum you need to just enter efp. You can use a third party FTP (File Transfer Protocol) software that runs on any platform like, Microsoft Windows, Mac-OS or Unix like systems, to transfer all the data you have collected so far to your lab computer. Here are the points to note for getting data by FTP: Do NOT use a secure FTP software. The FTP software that runs on NMR machines are anonymous FTP servers that have restricted access to only your data directories. Use a FTP software that has a graphical user interface. XWINNMR stores data in a complex tree structure and it will be very difficult if not impossible to use a text based FTP program to transfer all the directories successfully. An example of a FTP program with graphical interface is given below: 27

28 Left side of the display shows the local computer s disk. Right side shows the user data directories on the NMR computer: av301.chem.washington.edu While configuring the FTP software, use the connection Port number: 21. For username, enter: ftp For password enter: ftp Also, make sure that your FTP program is configured for passive mode of transfer. 28

29 APPENDIX A p1 p2 p3 p4 p6 LIST OF COMMONLY USED PARAMETERS 5 us to 12 us - 1H 12 us to 18 us - 13C F1 channel 90 o pulse width 10 us to 25 us - 1H 25 us to 40 us - 13C F1 channel 180 o pulse width 5 us to 12 us - 1H 12 us to 18 us - 13C F2 channel 90 pulse width 10 us to 25 us - 1H 25 us to 40 us - 13C TOCSY 90 1H pulse length; 25 us to 40 us calculated based on the p1 TOCSY 180 1H pulse length; normally derived internally from p6 p7 50 us to 80 us p us gradient pulse length variable - shaped pulse usually employed in water suppression schemes: WATERGATE, WET, water Flipback p11 p us ROESY spin lock soft pulse p us homospoil/gradient pulse length d0 d1 incremented delay in 2D relaxation delay : 1 to 5 times 1 to 1.2 s T1 J for CH: 125 to 222 Hz 1/2J d2 d3 1/3J d4 1/4J : used in HSQC delay for evolution of long range couplings d6 d7 d8 200 ms to 1 s NOESY mixing time d9 60 ms to 80 ms TOCSY mixing time d11 30 ms : default delay for disk I/O d12 20 us : default delay for power switching delay for inversion recovery delay for evolution after shaped pulse. d14 d to 500 us delay for gradient recovery PULSES DELAYS 29

30 d to 200 us delay for binomial water suppression in WATERGATE : 1/[2*d19] gives the position of next null cnst0 used in different contexts. cnst1 J (HH) cnst2 J (XH) cnst3 J (XX) cnst11 1 or 2 proton-proton J coupling stored as default ex: proton-carbon 1 bond J coupling ex: carbon-carbon 1 bond J coupling in labeled solute for multiplicity selection in DEPT or APT CONSTANTS cnst12 1 or 2 for multiplicity selection vc vd variable loop counter variable delay list VARIABLES l1 l2 l3 MLEV loop counter GARP loop counter loop for phase sensitive 2D or 3D using States or States-TPPI method l3 = td1/2 lb <line broad. Factor> for exponential window function td 4k to 64k number of FID points si 4k to 64k number of points in the spectrum LOOP COUNTERS MISCELLA NEOUS 30

31 ns 1 to td0 sw swh integer value ds 4 to 32 aq o1 ( proton) 10 ppm to 300 ppm 0.1 s to 5 s 1500 to 3000 Hz o1p (proton) 2.5 ppm to 7.5 ppm o1p (carbon) 75 ppm to 125 ppm o1p (fluorine) 0 ppm to -95 ppm o1p (phosphorous ) 0 ppm to 150 ppm o2 (proton) 1500 to 3000 Hz o2p (proton) 2.5 ppm to 7.5 ppm o2p (carbon) o2p (fluorine) o2p (phosphorous ) number of scans of a given experiment data is automatically written to disk after [ns/td0] scans. spectral width ppm units. Depends on the nucleus studied. spectral width Hz units. Depends on the nucleus studied and the spectrometer's base frequency number of dummy scans. Experiment is performed but the FID is not recorded length of FID in seconds. Solvents have long FID length. Offset of the spectrum center with respect to the base frequency (BF) of the spectrometer. Depends on the spectrometer's absolute base frequency. - CHANNEL F1 offset of the spectrum center in ppm units. - CHANNEL F1 Same as o1, but on CHANNEL F2 Same as o1p, but on CHANNEL F2 Same as o1p, but on CHANNEL F2 Same as o1p, but on CHANNEL F2 Same as o1p, but on CHANNEL F2 MISCELLANEOUS solvent pc 0.1 to 100 ensure that this is the same as the lock solvent you used, before starting the acquisition. peak picking sensitivity factor: pc < 1 will pick more peaks; pc > 1 will pick less number of peaks; pc = 1 is default 31

32 cy 10 to Height of the largest peak in cm. PH_mod rg phc0 0 to 360 phc1 0 to 180 pk : phase sensitiveps : power spectrummc : magnitude spectrum 1H : 1 to C (natural abundance) : 750 to 3000 depends on : solute concentration nucleus studied temperature zero order phase correction factor first order phase correction factor MISCELLANEOUS 32

33 APPENDIX B COMMAND NAME acqu ased eda edte getproso l go halt lock popt COMMONLY USED COMMANDS COMMAND ARGUMENT DESCRIPTION to switch to acquisition window show and allow editing of the limited set of parameters, relevant to the current experiment edit acquisition parameters in table format. start the temperature controller interface load the calibrated pulse and power levels for the current hardware start acquisition, but do not zero the contents of the memory buffer halt the acquisition after the current transient is completed. perform auto-locking of the spectrometer field, using the lock solvent signal (usually 2H) run the parameter optimization program automatically find out the optimal reciever gain rga rpar <parset> all to load an existing parameter set rsh <shim file> to load an existing shim file show stop tr wobb wpar proc <parset> all show the list of commands that are being executed by XWINNMR abort the acquisition immediately 'go' will not work after this transfer the transients collected so far to the disk tune and match the probehead to save the current dataset's parameters with a unique name write current shims to a unique shim file name wsh <shim file> xau <file> execute the automation program zg dir diro begin acquisition of the current experiment show the list of datasets recorded so far, for the current user show a list of valid users ACQUISITION FILE 33

34 re m n switch to a different [Exp. No, Proc. No.] combination of the current data set. m : experiment number n: processed data number wra wrp m n copy the raw data (FID) from the current experiment number to a different one. m: experiment number copy the current processed spectrum to a different proc.number within the same dataset n: processed data number FILE wrpa m n copy the current dataset in its entirety (including the FID and the spectrum) to a new [Exp. No, Proc. No.] combination. m : experiment number n: processed data number setti xwp open an editor for creating a title for the current dataset. launch XWINPLOT plot editor opens the editor, with the current dataset for display, as a default. PLOTTING abs perform the default baseline correction apk automatically correct the phase of the spectrum cfbsms edp efp ej em fid ft ij software reset the bsms system, when locking is not successful. edit processing parameters in table format. Perform FT after doing 'em' and apply the existing phase correction values i.e. phc0, phc1 eject the sample from the magnet apply an exponential window function to the FID, using the value of 'lb' to display free induction decay in the spectrum window Fourier transform the FID. inject the sample into the magnet PROCESSING maxi maximum threshold in cm for peak picking mi minimum threshold in cm for peak picking 34

35 pps display the picked peak list on screen. pscal global for peak picking sino sref winfunc calculate the signal to noise ratio you must define the signal and noise regions prior to this. automatically perform spectrum calibration based on lock solvent and TMS info. enter manual window function manipulation program PROCESSING 35