USER MANUAL FOR BASIC NMR ACQUISITION AND OPERATION OF BRUKER DPX400 and AV400 SPECTROMETERS

Transcription

1 USER MANUAL FOR BASIC NMR ACQUISITION AND OPERATION OF BRUKER DPX400 and AV400 SPECTROMETERS Ver 3.3 revised 2016 Contact: Dr. Alec Krunic Rm B72/ ext

2 CONTENTS INTRODUCTION.3 AVAILABLE INSTRUMENTATION 4 LABORATORY SAFETY AND SECURITY.5 ACCOUNTS AND LOGIN...6 DATA ACQUISITION AND PROCESSING.8 LOGGING OUT..12 DATA TRANSFER.12 OFF-LINE DATA PROCESSING.12 ACQUISITION AND PROCESSING OF 2D SPECTRA...13 UNATTENDED RUN OF A SERIES OF NMR EXPERIMENTS.17 XWINPLOT EDITOR 18 APPENDIX A..19 APPENDIX B..20 APPENDIX C..21 APPENDIX D..25 2

3 INTRODUCTION User manual is intended to be a beginner s guide for the acquisition of basic 1D and 2D spectra on spectrometers operating under Bruker XWINNMR software. It contains a step-by-step documentation of the acquisition protocols, it is not meant to be a comprehensive guide for advanced experiments. A more detailed version of the same can be found in the Bruker s XWINNMR software manual. One is expected to follow the rules and regulations of the NMR facility for the effective functioning of this facility. Rules and regulations of the NMR facility: Do s 1. In order to reserve instrument time, sign up on the sheet posted on the notice board across from 539 (MCP Main Office). Follow the posted instructions, also found on the Facility website. 2. The logbook has to be signed after every session with the instrument. Record any anomaly in the logbook. 3. While being in the Magnetic Resonance Facility (MRF), leave all metallic, electronic and magnetic appliances on the desk. 4. Submit your supervisor s memo for opening a new account. 5. Clean up after every use. 6. New users are encouraged to read any instruction posted in the facility. 7. Check if the sample volume is in accordance to the Sample Depth Gauge (SDG). Only clean NMR tubes should be loaded into the instrument. Don ts 1. Unauthorized access is strictly prohibited. 2. The spectrometer and computer settings should not be changed under any circumstance. 3. The height of the SDG should not be altered. 4. No personal notes, comments or complaints should be written in the logbook. 5. Smoking, littering and irresponsible usage of the facility will not be tolerated. 3

4 AVAILABLE INSTRUMENTATION Magnetic Resonance Facility (MRF) has two Bruker Avance digital NMR spectrometers located in Room B71S. Both systems were added in 2004 (9.4T/400 MHz Oxford magnets, 1999 and 2003 vintage). Selected features include: o Sample changers for 60 samples (BACS 60) on both 400s o Networked MS Windows -based computers with XWIN-NMR for acquisition off-line processing of the spectra o ATM (automatic tuning and matching) BBO probe on AV400 o Nitrogen evaporator for low temperature experiments on DPX400. All instruments are equipped with 5mm Z-gradient probe, in which inner coil is sensitive for X-nucleus and the outer coil is sensitive to proton. The default probe on the DPX is a QNP probe that can be used for detection of 13 C/ 31 P/ 19 F nuclei. The allowed temperature range on these probes is 150 o C to +150 o C. X- channel on the AV400 BBO (broadband observe) probe can be tuned to a variety of nuclei from 31 P to 109 Ag. Spectrometer is configured for elevated temperature experiments only. 4

5 LABORATORY SAFETY AND SECURITY NMR superconducting magnets contain large volumes of cryogenic liquid (helium and nitrogen). Except in rare instances the cryogens boil off over time at normal rates. In case there is loud noise followed with abrupt discharge of gases in the room, LEAVE THE ROOM IMMEDIATELY!!! New Magnetic Resonance Facility is equipped with oxygen sensors and low oxygen alarms located on the outside (orange strobe) and inside the B71 suite (audio signal/orange strobe). In the event the alarm is triggered, LEAVE THE ROOM IMMEDIATELY!!! WAIT OUTSIDE UNTIL THE ALARM IS CLEARED, AND/OR FOR CLEARANCE FROM THE FACILITY MANAGER (Dr. Krunic) BEFORE RE- ENTERING THE FACILITY. Magnetic objects are not allowed in the room, including pacemakers and medical implants, mechanical watches, cards with magnetic stripes, diskettes, coins and keys. Certain items may be permanently damaged. Safety tip: BEFORE APPROACHING THE MAGENT, LEAVE PERSONAL ITEMS ON THE BENCHES/DESKS. Moving magnetic material affects the magnetic field disturbing the measurements. Note: Magnetic field has no known harmful medical effects. NETWORK AND DATA SAFETY Processed data is removed on the regular basis from each workstation. Acquisition data is backed up periodically and eventually transferred to removable media upon user s departure from the department. Users may transfer their data for off-line processing, using CD-R and CD-RW media or network file share (see title Data transfer). USB devices are not supported because of security risks they may pose. 5

6 ACCOUNTS AND LOGGING IN Before logging in on any spectrometer, you must have a valid USER ID and password. A valid account and password can be acquired by: 1) submitting a memo from your supervisor to Dr. Alec Krunic and 2) filling out new user form (can be found on the Web at A secure password should be selected for your account. If a login fails three attempts, the account will be temporarily frozen for the security of your stored data. Login procedure for PC HP workstations running Microsoft Windows XP: SIGN IN the logbook before you start. <Ctrl><Alt><Del> to login or click the login window. o Type in your login-id (group ID) and password to begin. o click on XWIN-NMR 3.5 icon to start the NMR software Click in the command-line area. Remember, you can either use the buttons, or use the command-line equivalent is all cases. Insert the sample only if you hear the hissing sound of lifting gas. The pull-down menu or the commands can be used for operating the instrument. Users are expected to maintain the required codes of conduct in the facility at all times. This will help everyone to run the facility smoothly and efficiently. We also welcome suggestions from all users to help us work better. Immediately report any problem, or abuse of the instrument to Dr. Krunic. SAMPLE HANDLING Press the [Lift On/Off] button on the BSMS keyboard to eject the sample, and remove the sample from the magnet. o Note: Keep the rotor clean and clear of fingertip grease! Use Kimwipes provided, do not touch the spinner with bare hands. Check your tubes for any outside dirt. Insert the clean NMR tube into the rotor, and set the tube position as indicated with the 5/8 mm coil length marking on the depth gauge. o IMPORTANT: Use ONLY high-quality NMR tubes in the 400 MHz Wilmad Norell Kontes 535-PP-7 CONSULT FACILITY MANAGER 528-PP PP PP-8 6

7 o If you want to use tubes from other manufacturer consult the Facility Manager before purchasing. Insert the sample in the top of the magnet, then push the [Lift On/Off] button to insert the sample. Watch the sample, to ensure that it goes down (sometimes they get stuck at the top). Sample spinning is no longer necessary [SPIN] button should always be off 7

8 DATA ACQUISITION AND PROCESSING ACQUISITION AND PROCESSING OF 1D 1 H, 13 C, 31 P AND 19 F SPECTRA: The NMR experiments can be performed in the XWINNMR window. The following instruction is a stepwise protocol for acquiring proton 1D NMR spectra for the most common nuclei. Character ^ represents Enter key. (a) ej^ - ejects the standard sample. (b) To insert your sample in the probe, make sure your NMR tube is clean by wiping it with Kleenex tissues provided and push it gently into the sample holder. Use the SDG to adjust the tube height. Make sure you have sufficient sample ml is a good volume for a sample. DO NOT CHANGE THE SETTINGS ON THE SDG. Place the NMR tube in the sample port. (c) ij^ - inserts the sample into the magnetic field. NOTE: If a sample tube is broken inside the instrument, report to Dr. Alec Krunic immediately. (d) edc^ (or new^) - edit current data set. XWINNMR allows for the storage of your data according to the format in the window that opens up upon this command. This will be possible only if there is an existing file for you where you can store your data. If you have an account, you are most likely to have a data set with your login name. In case the computer can not read your data set contact Dr. Alec Krunic. Choose your file name and expno appropriately. Click SAVE to confirm your choices. (e) rpar^ - reads parameter file. A parameter file is a set of instrumental parameters for running an experiment. (f) Select +PROTON_16, representing the parameter file for running 1 H experiment with 16 scans. A new window opens up. Click on the copy all button to loadi all the required parameters. Similarly, other experiments with + prefix can be selected followed by copy all. Experiment lists for all spectrometer are attached in Appendix A. 8

9 Changing the solvent and locking: (g) eda^ - edit acquisition parameters. This loads the acquisition parameters which can be changed to run your experiment. You will have to scroll down and find a toggle button SOLVENT and choose the solvent for your sample. You will also have to find the PROSOL button and click on it to set it to TRUE. (h) Once you have made your choices for the experimental parameters, you have to click the SAVE button to update the parameters and also exit that window. (i) lockdisp^ - Displays the lock signal. A new window will open up which shows a plot of lock level and noise in the solvent signal. This window is a guide for effectively shimming the instrument. Shimming: (j) rsh^ - recall shim files. A part of our efforts in maintenance goes into shimming the NMR instruments. We save the shimming parameters as shim files for the best solvent spectra. They are frequently updated. Less common solvent can be added at a user s request. (k) Select the latest shim file for your solvent (e.g. if your solvent is CDCl3 LATEST_CDCl3 is the name of the corresponding shim file). One cannot assume that the shims from the previous user are fine. (l) lock^ - auto locks the instrument. You will have to select the correct solvent when you lock. NOTE: Check Lock Field, Lock Phase, Lock Gain on the BSMS keyboard before proceeding. (m) Shimming procedure: If you are familiar with manual shimming you can shim the instrument for better line shape and resolution. The correct order is to shim Z1 Z2 Z1 X and Y [press ON AXIS button, and then Z button. For off axis shims press the corresponing button then Z0 ]. Then you can shim for XZ and YZ followed by X and Y, again. All the time you are looking for the highest lock level. If the lock level goes beyond scale, bring down the signal by reducing lockgain. A good lock level is anywhere between the second and third row from the top. Once the lock level has been set you can fine tune the instrument by optimizing Z1, Z2 or Z3. Here you are trying to reduce the noise in signal. Proceed with gradient shimming by typing gradshim^, then first clicking on 1D2H radio button in the control panel that opens up and then on Start Gradient Shimmint button. See Appendix D. NOTE: 1D-gradshimming corrects only Z-shims. It is highly recommended to correct X- and Y- shims manually before repeating the procedure or starting data acquisition. (n) rga^ - Adjusts receiver gain. Makes sure the FID received by the instrument is not saturated. (o) rg^ - Note the value. It gives an idea about concentration of your sample. (p) ns^ - number of scans. Allows you to select the number of scans for your acquisition. It has to be a number in the series of 2 n. 9

10 (q) expt^ - Gives the experiment duration. You can adjust the number of scans accordingly. (r) zg^ - Starts the experiment. Once you have acquired a 1 H NMR spectrum, you have to follow the same set of instructions for starting a 13 C or 31 P or 19 F experiment. You can do that by setting a different experiment number with the edc^ command to create a new experiment. Otherwise, you will overwrite on an existing FID and lose your data permanently. (s) setti^ - Sets title. You can keep a record of any experiment and its particulars for future referencing. (t) lb 1.0^ - Sets line broadening to 1.0Hz (3.0 Hz for carbon (u) ft^ - Fourier transforms the FID (v) Phasing - Click PHASE button on the screen. A new display panel appears with PH0 and PH1 buttons. Press on PH0 (zero order phase correction) with left mouse button, hold and movethe mouse up or down to make the baseline of the spectrum straight. You can also expand sections of the spectrum and increase the peak heights to make it easier to phase the spectrum. Next, use the PH1 for phasing the peaks to make them look symmetrical. Once this is achieved, press RETURN. A new window opens up with a few options. Press SAVE AND RETURN if you are happy with the phasing. If you want to start over again press RETURN. (w) efp^ - This command helps you to reduce the noise in the baseline. Alternatively you can type ef^ followed by apk^ at step (v) and (w) and proceed with manual phasing. Integration, Peak picking and Plotting in XWINNMR in UNIX/WINDOWS: The mouse for UNIX has three buttons. Left (L), Middle (M) and Right (R). L click helps in scanning the Spectra by putting an arrow indicative of the position in the spectra. M click helps in selecting regions in the spectra. In normal mode if you select a region in the spectra using the M button, it is expanded. To unbind the cursor from the M click, you need to L click. PC mouse has a wheel instead of the third button. (a) Integration - Click on the INTEGRATE button. This will put the spectra in the integration mode. (b) Adjust your spectra such that you have a clear view of the peaks to be integrated and the peaks are well spaced out. You can use the navigating buttons to move from one peak to another incase the peaks of the entire spectra do not fit in the window. (c) L click to display the cursor on the spectra. (d) M click selects the left point and another M click selects the right point of the region to be integrated. You can follow this on all the peaks of interest. (e) To calibrate the integration, you can L click to unbind the cursor and point the cursor to the integral of interest and L click. Press CALIBRATE button and enter any number from

11 (f) If you are satisfied with the integration press RETURN and select SAVE INTEGRATION AND RETURN. Otherwise, you can press return and integrate again. If you want to deselect a single integral double L click on the peak and select DELETE FROM CURRENT. (g) You can calibrate the spectrum to TMS reference or solvent peaks anytime to the appropriate ppm. Save and return. (h) edg^ - Gives the option to customize display parameters like integral peak labels, plot units etc. (i) Once you are satisfied with your display parameter settings, click SAVE. (j) Peak picking - If you do not want peaks, which are too tall, and too short (like impurities and solvent) you can set the threshold based on peak height. MI^ for minimum peak height (0 or greater) and MAXI^ for maximum peak height. pps^ command will print a pick list on screen in both Hz and ppm. Plotted spectra will only contain one or the other. Use YU button to establish peak height in cm units. You may need to run xwp_pp^ command if the peak labels do show up in the spectrum. (k) view^- Shows the spectrum as it is going to be printed. If you are not satisfied with it, follow the steps from (h) (k) until you have what you want. (l) plot^ - prints the default spectrum window. Use dpl1^ command or DP1 button to define the spectrum window of your choice. Re-scale the spectrum with cy^ (default value is 19) and re-plot, if the peak intensity is too low. NOTE: The buttons in the user panels are interactive. On pointing the cursor to any button on the display panel, information about its purpose and usage will show up at the bottom of the screen. Recording Standard 13 C, 31 P and 19 F 1D spectrum on DPX400: 1. edc^- change EXPNO. different from the proton. Usually rpar^ - choose +C13CPD or +P31CPD or +F19CPD parameter file. CPD spectra are acquired with decoupling of protons, mostly. 3. The rest of setup is exactly the same as for 1D proton spectrum. 4. Following ft^, you may use apk^ (auto phase correction) then efp^ 5. abs^ (to correct the baseline if necessary) 11

12 LOGGING OUT: Before you logout, replace your sample with standard sample provided (1:1/H2O- D2O). Create a new experiment folder, e.g. lock, and load proton experiment. Change solvent to D2O or D2O+H2O and lock on the solvent. Proceed with exiting and logging out. DATA TRANSFER: Your data is saved to D:/data/username/nmr or in certain cases to C:/Bruker/XWINNMR/data/username/nmr Data can be burned on a CD/CDRW on each NMR workstation. Flash memory drives are not permitted for security reasons. MRF workstations are linked to a network share (NMRFILESHARE, W:\). The share can be accessed through offline processing workstation (HP1100\DATASTATION) in Room B73. For login information contact Dr. Krunic. To move data for offline processing or backup, go to your data folder on NMR workstation, select data you want to transfer and paste it into NMRFILESHARE/data/username folder. OFFLINE PROCESSING: (a) Start XWINNMR or XWINPLOT on DATASTATION (b) Locate your NMR data by clicking on File menu, then Search option. Your data is either on W:/ or on G:/. Append the dataset you wish to open and click Apply. Data in the form of NMR spectrum or FID will appear in the window (c) You can also type new^ in the main XWINNMR window, and change DISK, USER, FILE and EXPERIMENT NUMBER fields accordingly (d) Network printing is enabled to the network HP5100 printer. 12

13 ACQUISITION AND PROCESSING OF 2D SPECTRA Before starting an acquisition it is important to achieve optimal spectrometer conditions. Tube and solvent quality is also important, refer to section on Sample Handling. (a) Check the temperature of the probe by typing edte^, wait until the control panel starts up and verify that you are operating at 298K (the default temperature). Exit the control panel by clicking on File menu, then choose Exit. (b) Tune the probe to ensure maximum sensitivity for detection. Tuning/matching is automated with the ATM BBO probe on AV400. Other spectrometers are equipped with probes that do not support manual tuning. First, check the tuning by typing wobb^, wait until you see the message wobb curve displayed in acquisition window then type acqu^. Vertical blue line marks the spectrometer frequency. The tuning profile is display as a dip at the actual resonant frequency. Your goal is to match this dip with the vertical line. In order to do this type, type atmm^ and wait for messages and atmm control panel to open up. [refer to Appendix C for screenshots of wobb/atmm windows] (c) [Alternative approach: From the wobb window type stop^ and return^ to go back to main XWINNMR window. Type atmm in the command line to call the auto tune and match control panel, which also calls the same wobb routine, and type acqu^ again. You should see the curve identical to one observed with the wobb command.] (d) Atmm control panel has two lines with single/double/triple arrows defining the rate at which the motor turns tuning/matching rods. DO NOT USE TRIPLE ARROWS, as you can seriously damage the probe!!! Click on the arrow button, wait for response, then click again, starting with tuning then proceed to matching. When the curve lines up with the vertical line, click on File then Exit (answer No to the question about saving the current position). If you are doing a heteronuclear experiment, you will also need to tune the other nucleus (usually 13 C). This can be done either by setting up a 13 C observe experiment and repeating the procedure above, or by waiting until you set up your 2D experiment. In that case, wobb^ command will display the curve for other nucleus first; in order to observe proton you will need to type wobb f2^. Standard set of 2D experiments on both 400 MHz spectrometers include: +gcosy, +gdqfcosy, +gtocsy, +gnoesy, +groesy, +ghmqc, +ghsqc, +ghmbc. Non-gradient variants are also available (check Appendix A for detailed information). All the 2D acquisition parameter files come with a preset spectral widths, covering most common region of proton chemical shifts( 0-16 ppm for 1 H, 0-160/220 ppm for 13 C). Hence, user should adjust the spectral width according to proton spectra of the sample of interest, maximizing resolution in t1 dimension. Relative order of sensitivity of 2D experiments: TOCSY>DQF-COSY>HSQC, HMQC>HMBC, NOESY, ROESY 13

14 Data acquisition is similar to proton. Processing could be different for some 2D experiments. xfb^ command automatically processes and phases most common experiments except for the phase sensitive ones. Steps in any 2D data acquisition: (a) Check operating temperature as described above (b) Tune and match, if necessary (on the ATM probe only) (c) Shim on the sample (manual/autoshim/gradient) (d) Collect survey 1D proton spectrum with ns=8, ds =0 o1p=6 ppm, sw=20 ppm (e) Calibrate proton high-power 90 degree pulse (PW90) (f) Reacquire 1D proton spectrum using found PW90 (g) After phase correction and efp^ the spectral region has to be identified. This will be the region where the resonances are present. (h) Click on sw-sfo1 button on the screen. (i) The values for sw and o1 are used for the 2D setup. (j) Reacquire 1D proton spectrum which can be used a F2 projection (k) rpar^ and read a 2D experiment parameter file (l) eda^ and set PROSOL to True. Verify that TD parameter for the F2 dimension is set to 2048/1024, in particular for heteronuclear experiments which use 13 C decoupling. Next to it is the value for F1 dimension. Select values of 128/256/512 for low/medium/high resolution. NOTE: experiment time will increase when you increase this value. (m) sw<value determined by 1D>^ (n) o1p<value determined by 1D>^ (o) rga^ (p) zg^ (q) wait for 1 st increment to complete (r) rser1^ (rser2^ for TOCSY) (s) fp^ (t) phase the 1D spectrum and SAVE. (u) You need the phc0 and phc1 values after phasing. (v) acqu^ (w) stop^ (x) edp^ (y) set the phc0 and phc1 values (See Appendix B for information how to phase 2D spectra). (z) zg^ Processing magnitude mode experiments (e.g.: COSY, HMBC) (a) xfb^ (b) syma^ - to symmetrize a spectrum (optional, for COSY only) (c) Select plotting region. This can be done in two ways: (1) Move the mouse over the spectrum window and click the left mouse button. A crosshair will appear on 14

15 the spectrum. Move the crosshair with the mouse to the lower/upper left edge of the region you want to expand. The middle mouse button will then freeze the crosshair in this location. Moving the mouse again will move a second crosshair; move this to the upper/lower right edge of the region you want to expand and then click the middle mouse button again. Clicking the left mouse again button will release the cursor from the spectrum. This will leave a box on the spectrum showing the desired expansion area. (2) Move the mouse to the left border buttons and click on the button close to the top that has the rectangle on it (this is the right-hand button on the fourth row down from the top). This will expand the selection region of the 2D spectrum. To go back to the full spectrum, click on the ALL button close to the expansion button; the EXP button will re-expand to the previously chosen region. Alternatively, click on the button with rectangle on it, and then use mouse pointer as a lasso to create a box around part of the spectrum you are interested in. (d) Define plot region with defplot^. Answer y if you want contour plot. Adjust vertical scaling to desired level. (e) Add projections using graphics editor. Type edg^, look for paramaters PROJ1 and PROJ2 and EDPROJ1 and EDPROJ2. Projections should be enabled by the default. (f) Click on ed button next to EDPROJ1 or 2. In new window change fields for DISK, USER, FILE and EXPERIMENT NUMBER. Click on SAVE then SAVE again. (g) view^ to check the spectrum layout (h) plot^ Processing phase sensitive experiments (e.g.: NOESY, HSQC). For detailed description see Appendix B. (a) rser 1^ (rser 2^ for TOCSY) (b) sinm^ (c) ft^ (d) phase 1D. Then click SAVE AS 2D AND RETURN. (e) Select 2D button. (f) edp^- change processing parameters if required. (g) xfb^ (h) click on the PHASE button (i) Choose COLUMNS, phase, type xf1p^ (j) Phase rows and proceed with xf2p^. Phase columns and rows as necessary. (k) Click +/- to display both negative and positive peaks. (l) syma^ - to symmetrize the spectrum (optional for TOCSY, NOESY/ROESY). (m) Select plotting region (n) Define plot region with defplot^. Answer y if you want contour plot (o) Add projections using graphics editor. Type edg^, look for paramaters PROJ1 and PROJ2 and EDPROJ1 and EDPROJ2. Projections should be enabled by the default. 15

16 (p) Click on ed button next to EDPROJ1 or 2. In new window change fields for DISK, USER, FILE and EXPERIMENT NUMBER. Click on SAVE then SAVE again (q) view^ to check the spectrum layout (r) plot^ 16

17 UNATTENDED RUN OF A SERIES OF NMR EXPERIMENTS This procedure enables you to run several experiments without having to start each experiment one by one. It is extremely useful for overnight/ weekend runs. (a) Prepare for running a NMR experiment similar to 1D proton spectrum (LOCK and Shim etc.) (b) new^ (c) Type dataset name under NAME (d) Type experiment number. (e) SAVE. (f) rpar^- select first experiment from pulse sequence library. (g) eda^ - Choose solvent, PROSOL:TRUE etc. (h) SAVE (i) rga^ (j) ns^ - type number of scans. (k) expt^- note experiment time. (l) new^ (m) Increase experiment number by one. DO NOT change dataset name. (n) SAVE (o) Continue steps 6-11 for each new data set. (p) Calculate the total length of time for all experiments. Alternatively, you can use multiexpt command to obtain total running time: re 1^, then multiexpt^, enter number of experiments. (q) Change number of scans as time permits. Use re (expt. no.)^ for switching between experiments. (r) re 1^ (s) type multizg^ (t) Type the total number of datasets you plan to run and press enter. This starts the run. 17

18 XWINPLOT XWINPLOT is Bruker s external plot editor for creating high quality printouts of NMR data. Version 3.5, installed on Windows based workstations, allows export of NMR data in graphical (JPEG, TIFF), Adobe PDF and Microsoft (WMF, EMF) file formats. (a) xwp_pp^ - create peak picking file (b) xwp^ - start XWINPLOT on all platforms. (c) Prepare the plot as you please using Edit menu. Alternatively you may open one of the 1D or 2D plot templates (.*xwp files) and update with current data. (d) Go to FILE menu and click PRINT. For more details and training how to use this software, contact MRF TA. 18

19 APPENDIX A LIST OF COMMONLY AVAILABLE NMR EXPERIMENTS ON BRUKER DPX AND AV400 The following parameter sets have standard parameters saved, their names starting with a plus sign prefix to distinguish them from the Bruker s parameter sets. C13_DEPT135 C13_DEPT45 C13_GATED_DEC C13_INV_G_DEC CARBON13_CPD COSY45SW DQF_gCOSY F19_1Hdcoup HMBC_nongrad P31CPD PROTON_16 PROTON_GARPd_C PROTON_WATSUPP TOCSY_2D gcosy ghmbc ghsqc gnoesy groesy EXPERIMENTS AVAILABLE ONLY ON AV400 ghsqc-tocsy PROTON/TOCSY/ROESY with solvent ghsqc_hs suppression using excitation sculpting ghsqc_ed ghsqc-tocsy_ed ghmbc phase senstitive g1,1-adequate sel1d_tocsy/noesy/roesy 15N-versions of HSQC and variants 15N INEPT for direct detection of 15N PULSE PROGRAMME ABBREVIATIONS USED BY BRUKER Some common abbreviations are listed below. Pulse programme name can be checked by entering pulprog^ in the command line or going into acquisition control screen (eda^). More details can be found in the pulse programme file itself (edcpul^): qf magnitude acqu mode ph phase sensitive acqu mode lp lowpass filter gp/gs gradient experiment pr presaturation et Echo/Antiecho lr long range df/mf quantum filtered l9 WATERGATE nd not decoupled sel - selective mlev, dipsi, dip TOCSY es excitation sculpting si sensitivity enhanced sp using shaped pulses 19

20 APPENDIX B PHASE CORRECTION PROCEDURE FOR 2D PHASE-SENSITIVE EXPERIMENTS FOR BRUKER XWINNMR SOFTWARE VERSIONS 2.x/3.x Before you begin: Generally in a 2D spectrum, F2 dimension (rows) is phasecorrected first, and then the F1 dimension (columns). To phase correct the spectrum in F2, three rows each with a cross peak should be selected. The cross peak of one row should be to the far left of the spectrum, the cross peak of the second row should be close to the middle, and the one of the third row should be to the far right of the spectrum. Enter the phase correction menu by clicking on the button. Select one row by clicking on with the left mouse button, a cursor will apear in the 2D spectrum window located in the upper left corner of the display. Move the mouse until the horizontal cross hair is aligned with a row that has a cross peak. Select the row by clicking the middle mouse button. If the selected row does not intersect the most intense portion of the cross peak, click with the left mouse button until it does. Once the desired row is selected, click on with the left mouse button to move the row to window 1 appearing in the upper right hand corner of the display. Repeat the selection of rows described above for a row with a cross peak in the middle and another row with a cross peak at the right edge of the spectrum and move them to window 2 and 3, respectively. Click on the or the button to tie the cursor to the biggest peak of the row in window 1. Phase Correct this row using the 0 th -order phase correction button. Correct the 1 st -order phase correction for the other two rows using the button and observe the rows in window 2 and 3, respectively. Save the phase correction by returning to the main window (select Save & return at the prompt). NOTE: To phase correct the spectrum in F1, repeat the above procedure by selecting three columns rather than rows. 20

21 PROBE TUNING AND MATCHING. APPENDIX C In order to observe probe tuning you need to type in wobb^, then acqu^ and wait for the curve to appear. From this observed window you can call up autotune and match control panel by typing atmm^ (see Appendix D), in case you need to adjust tune and match. From the same window, you can check the tuning for other channels by typing wobb f2^. NOTE: wobb curve for X-nuclei may appear slightly different from the 1H curve. Stop the acquisition with stop^ command, and exit the window by typing return^ or clicking on the return button. In case you are unable to observe a wobb curve, contact the MRF Manager. Following pages contain examples (1-4) of wobb curves in respect to tune and match settings. 21

22 1) 2) 22

23 3) 4) 23

24 AUTOTUNE AND MATCH UTILITY FOR ATM ENABLED PROBES. Atmm control panel has two lines with single/double/triple arrows defining the rate at which the motor turns tuning/matching rods. DO NOT USE TRIPLE ARROWS, as you can seriously damage the probe!!! Click on the arrow button, wait for response, then click again, starting with tuning then proceed to matching. When the curve lines up with the vertical line, click on File then Exit (answer No to the question about saving the current position). If you are doing a heteronuclear experiment, you will also need to tune the other nucleus (usually 13 C). This can be done either by setting up a 13 C observe experiment and repeating the procedure above, or by waiting until you create your 2D experiment dataset. In that case, wobb^ command will display the curve for X nucleus first; in order to observe proton you will need to type wobb f2^. 24

25 APPENDIX D GRADIENT SHIMMING UTILITY IN XWINNMR 3.5 AND TOPSPIN 1.X/2.X Your username will be displayed here 1d2h.bbo is the filename displayed in this box when gradient shimming option is set up. 25