CONTENTS 1D EXPERIMENTS D EXPERIMENTS APPENDIXES... 83

Transcription

1 Jari Koivisto, Ph.D. Aalto University School of Science and Technology Faculty of Chemistry and Materials Sciences Department of Chemistry Bruker Avance DPX400 User Manual Basic and Advanced 1D and 2D Experiments (Version 1/2010)

2 CONTENTS 1 Introduction NMR Facility Policies Preparing For Acquisition Leaving the Spectrometer D EXPERIMENTS H NMR C NMR DEPT 45, DEPT 90, and DEPT H, 1 H Correlation: Selective 1D COSY Extended 1 H, 1 H Correlation: Selective 1D TOCSY H, 1 H Correlation Through Space: Selective 1D NOESY, ROESY, and off-res-roesy Highly Selective Pseudo 2D Experiments: CSSF-TOCSY, CSSF-NOESY, and CSSF-off-res-ROESY Calibration of the DPFGSE Sequence Direct 1 H, 13 C Correlation: SELINCOR F NMR P NMR D EXPERIMENTS H, 1 H Correlation: COSY H, 1 H Correlation: DQF-COSY Extended 1 H, 1 H Correlation: TOCSY H, 1 H Correlation Through Space: NOESY and gs-noesy H, 1 H Correlation Through Space: ROESY, T-ROESY, and off-res-roesy Direct 1 H, 13 C Correlation: HMQC Direct 1 H, 13 C Correlation: HSQC Long-Range 1 H, 13 C Correlation: HMBC Long-Range 1 H, 13 C Correlation: CIGAR-HMBC Long-Range 1 H, 13 C Correlation: G-BIRD-HSQMBC APPENDIXES Appendix A: Cleaning Procedures for NMR Sample Tubes Appendix B: Some Useful Commands and Tips Appendix C: About NOE Experiments Appendix D: Tuning and Matching the Probe Appendix E: Linear Prediction Appendix F: Variable Temperature Experiments... 97

3 2 1 Introduction This manual provides systematic instructions on how to perform basic and advanced one- and two-dimensional NMR experiments on Bruker Avance DPX400 spectrometer located in the Laboratory of Organic Chemistry at the Aalto University School of Science and Technology. The following 1D and 2D NMR experiments are included: 1D: 1 H, 13 C ( 1 H decoupled), DEPT 45, DEPT 90, DEPT 135, 1D COSY, 1D TOCSY, 1D NOESY, 1D ROESY, 1D off-res-roesy, CSSF-TOCSY, CSSF- NOESY, CSSF-off-res-ROESY, DPFGSE, SELINCOR, 19 F ( 1 H coupled and decoupled), and 31 P ( 1 H coupled and decoupled) 2D: COSY, DQF-COSY, TOCSY, NOESY, gs-noesy, ROESY, T-ROESY, offres-roesy, HMQC, HSQC, HMBC, CIGAR-HMBC, and G-BIRD-HSQMBC Additional information can be found in Appendixes A F at the end of this manual. Note that this manual is not intended as a textbook on NMR theory or spectrum interpretation. See section 1.5 for recommended reading on these subjects. 1.1 The instrument Some features of Bruker Avance DPX400 spectrometer include: 9.40 Tesla standard bore UltraShield magnet B-VT 3200 variable temperature unit GRASP II Z-gradient accessory QNP probehead (5 mm) with Z-GRASP coil; automatically switchable between nuclei ( 19 F, 31 P, 13 C/ 1 H) Bruker Orthogonal Shim System (BOSS I) with 20 shim gradients Two Frequency Control Units (FCU ) 16/18 bit SADC for an effective spectral width up to 150 khz NMR Case automatic sample changer for 24 samples Windows XP workstation with the TopSpin 1.3 software package 1.2 The TopSpin Interface The TopSpin window consist of several areas, bars, fields and buttons (Figure 1): 1. The Title bar with program version and computer name 2. The Menu bar with pull down menus for TopSpin operations 3. The Upper Toolbar with buttons for data handling, switching to interactive modes, display settings, and starting acquisition 4. The Lower Toolbar with buttons for display manipulations 5. The Browser and Portfolio for browsing, selecting, and opening data 6. The Command Line for keyboard input 7. The Status bar for the display of information during command execution

4 3 8. The Acquisition Status bar from where the acquisition can be followed and controlled 9. The Data area 10. The Lock display Magnet and security Figure 1. The TopSpin window Large attractive forces may be exerted on magnetic materials or equipment in proximity to the NMR magnet system, which is always at field. The force may become large enough to move the equipment uncontrollably towards the NMR magnet system. Small pieces of equipment (e.g. tools, bolts and nuts) may therefore become projectiles. Large equipment (e.g. gas bottles) could cause bodies or limbs to become trapped between the equipment and the magnet. The closer to the magnet system, the larger the force. The larger the equipment mass, the larger the force. Due to the very effective shielding of the superconducting coil, the effects of the magnetic stray field are minimized. Nevertheless, keep in mind that directly above and

5 4 directly below the magnet the stray field is very high and the attractive forces on magnetic items are very strong! The operation of medical electronic implants, such as cardiac pace makers, may be affected by the magnetic field. Other medical implants, such as aneurysm clips, surgical clips or prostheses, may contain ferromagnetic materials and therefore would be subjected to strong attractive forces near to the NMR magnet system. Items such as watches, tape recorders, and cameras may be magnetized and irreversibly damaged. Information encoded magnetically on credit cards, diskettes and magnetic tapes may be irreversibly corrupted. Note that moving magnetic materials affects the magnetic field disturbing the measurements. Note also that a strong knock against the magnet may result in the unlikely event of the magnet quenching or of a cryogenic failure, where up to 100 m 3 of helium gas may evolve over a period of several minutes. Personnel should evacuate the area in such a situation since helium gas can displace oxygen in the room. A quench warranting evacuation would be obvious by the noise of the erupting gas and clouds of vapor. DO NOT BRING ANY MAGNETIC OBJECTS NEAR THE MAGNET! BEFORE GOING NEAR THE MAGNET, EMPTY YOUR POCKETS AND TAKE OFF YOUR WATCH! 1.4 Notation In this manual different font styles and symbols are used to represent various types of information: Arial is used for buttons, menus, and icons to be clicked Bold Arial is used for the TopSpin commands to be entered on the command line Italics Times New Roman is used for pull down menu commands Underline is used for the web site addresses Expressions in [SQUARE BRACKETS] and in bold capitals represent keys on the BSMS keyboard (the BSMS keyboard is the gray rectangular pad with buttons, a control wheel, and an LED display sitting next to the computer) 1.5 References and recommended reading References: Avance Beginners Guide TopSpin Bruker Avance 1D and 2D Course TopSpin Users Guide TopSpin Acquisition Reference Guide TopSpin Processing Reference Guide TopSpin 1.3 Installation Guide for Windows XP

6 5 Above-mentioned manuals can be found on Bruker BioSpin website Note that in order to access these documents, you will need to order a free account for Bruker NMR WWW Server. User manuals can also be found under the Help menu in the TopSpin window. Note also that manuals and a lot of other useful information can be found on the NMR Guide website. NMR Guide is a WWW-based training, teaching and practical tool aimed to everybody interested in NMR spectroscopy. NMR Guide can also be found under the Help menu in the TopSpin window. Recommended reading: H. Günther, NMR Spectroscopy: Basic Principles, Concepts, and Applications in Chemistry, 2 nd ed., Wiley, F. J. M. van de Ven, Multidimensional NMR in Liquids, Basic Principles and Experimental Methods, Wiley-VHC, H. Friebolin, Basic One- and Two-Dimensional NMR Spectroscopy, 3 rd ed., Wiley-VHC, T. D. W. Claridge, High-Resolution NMR Techniques in Organic Chemistry, Tetrahedron Organic Chemistry Series Vol. 19, Pergamon, D. Neuhaus, M. P. Williamson, The Nuclear Overhauser Effect in Structural and Conformational Analysis, 2 nd ed., Wiley-VHC, M. H. Levitt, Spin Dynamics: Basics of Nuclear Magnetic Resonance, Wiley, S. Berger, S. Braun, 200 and More Basic NMR Experiments, A Practical Course, Wiley-VHC, J. Keeler, Understanding NMR Spectroscopy, Wiley, 2005.

7 6 2 NMR Facility Policies Users not following the rules will lose their privileges to use the instrument! 2.1 General policies Only authorized users may operate the instrument in the NMR facility. Use only your own dataset. Using someone else's dataset is strictly forbidden Authorized users are persons who have received instruction from the NMR Facility Manager and then have proven to be able to operate the instrument. Spectrometer training is provided on request basis Use only those parameter sets that begin with AN_! No tools, test equipment or manuals are to be removed without the permission of the Facility Manager Standard library pulse sequences, parameter files, system configuration files, standard AU programs etc. are never to be modified except under direct instruction of the Facility Manager Do not make any changes in the hardware configuration In order to avoid disk space problems, backup all unnecessary data and then delete it Do not bring drinks or food in the NMR room Do not wear gloves while operating the spectrometer Do not use internet or on NMR PC (minimize virus threats) Sign the NMR logbook before leaving the room Door should be shut (and locked) when you leave the room All unusual spectrometer behavior should be noted in the logbook and reported to the NMR Facility Manager as soon as possible 2.2 Reservation rules Reservation list can be found at X:\Booking lists etc\nmr\nmr BOOKING.xls The previous day is the earliest day for reservation. Daytime (8:00 18:00) from Monday until Friday the maximum reservation time is 2h. Long measurements should be done during nights or weekends. Overnight runs start earliest at 18:00 and finish latest at 8:00 a.m. Reserve only one blue slot per every uneven hour Remember that variable temperature experiments require you schedule enough instrument time to bring the probe back to room temperature when you are finished If you do not use the whole time you have booked, please inform the next one on the list. If the instrument is free, the fastest one may use it Do not delete reservations made by others. If there is something to complain about someone s reservation, consult the NMR Facility Manager The NMR Facility Manager reserves the authority to make exceptions to these rules when it is necessary or desirable to do so!

8 7 3 Preparing For Acquisition 3.1 Sample preparation and positioning The sample quality can have a significant impact on quality of the NMR spectrum. The following is a brief list of suggestions to ensure high sample quality. Sample preparation: Use deuterated solvents whenever possible For 1 H NMR, 1 5 mg of the sample is sufficient. For high molecular weight samples, more concentrated solutions are sometimes recommended. However, too concentrated solution leads to lower resolution due to saturation and/or increased viscosity. For 13 C NMR ca. five times the concentration is recommended For 2D measurements, the sample should be concentrated enough to achieve an acceptable S/N ratio. As a rule of thumb, 25 mg of the sample is sufficient for nearly everything, including 1 H, 13 C HMBC experiment. With samples of only 1 5 mg, the homonuclear 1 H, 1 H experiments (e.g. COSY) are still feasible, but those involving 13 C may take overnight to complete Always use the same sample volume or solution height. This minimizes the shimming that needs to be done between sample changes. The recommended value is (at least) 0.6 ml or 4 cm solution for 5 mm sample tubes Always use clean and dry sample tubes to avoid contamination of the sample. See Appendix A for proper cleaning procedures for NMR sample tubes. Brand new NMR tubes straight from the packet are not usually very clean! Always use high quality sample tubes to avoid difficulties with shimming (e.g. Wilmad 527-PP or 528-PP) Filter the solution. A small plug of fresh medical cotton wool at the neck of a Pasteur pipette will do the trick. However, it is necessary to pre-rinse it with a little amount of the solvent to be used to flush out any loose fibers Sample positioning: Use the sample depth gauge to position the sample tube in the spinner. Be careful with the depth gauge. Do not change the depth! The slider should be in position 5 15 mm Always wipe the NMR tube clean before inserting it into the spinner. Wipe it once more after the sample depth has been adjusted Sample tube depth adjustment: First, seat the spinner on top of the depth gauge. Second, carefully push the sample tube through the spinner until the bottom just touches the top of the slider. Remove the depth gauge before inserting the sample and spinner into the magnet! For experiments using sample spinning, be sure that the spinner, especially the reflectors, is clean. This is important for maintaining the correct spinning rate. Do not touch the spinner with your bare hands! Check that the sample tube is tightly held in the spinner so that it does not slip during an experiment

9 8 3.2 Open the NMR software If the workstation is locked, press Ctrl+Alt+Delete and then enter password when prompted (User name: nmr) If the software is closed, double-click the TopSpin 1.3 icon In order to display data from the Browser, proceed as follows: 1. Expand D:\Bruker\TOPSPIN (double-click or click the + button) 2. Expand your folder 3. Left-click-hold the desired item and drag it into the data area 4. Select the desired expno/procno combination and click on 3.3 Insert the sample Press [LIFT ON-OFF] to eject the old sample Change the samples Press [LIFT ON-OFF] to send the new sample down The DOWN indicator in the BSMS keyboard will light up as soon as the sample is safely in the probe Check that the spinner is on (a lit LED on the [SPIN ON-OFF] button) 3.4 Lock If the lock window is closed, click on (lockdisp) (lock) Select the appropriate solvent from the menu and click on Wait for the lock: finished message NOTE: If a sample has been replaced with one using the same solvent, then the system will usually be locked automatically. 3.5 Shim on the lock signal Adjust Z and Z 2 iteratively until the maximum lock level is found: 1. *Optional* Press [LOCK PHASE] and turn the control knob until the lock signal is maximized 2. Press [Z 1 ] and adjust the Z 1 shim to maximize the lock level 3. Press [Z 2 ] and adjust the Z 2 shim to maximize the lock level 4. Repeat steps 2 and 3 until the maximum lock level is found; always end with the Z 1 shim. The lock signal should now be at the upper third of the lock display 5. *Optional* Press [LOCK PHASE] and maximize the lock level 6. Finally press [STD BY] *Optional* Adjust X and Y iteratively until the maximum lock level is found (as described above for Z and Z 2 ). These adjustments should be done only if an experiment without sample spinning is to be performed:

10 9 1. Press [SPIN ON-OFF] to stop spinning 2. X: First press [X], then [Z 0 ], and then adjust the shim value 3. Y: First press [Y], then [Z 0 ], and then adjust the shim value 4. Optimize Z and Z 2 as described above, but this time without sample spinning NOTE: If the lock signal goes off-scale, press [LOCK GAIN] and adjust the gain (turn the control knob) to bring the lock signal back to the upper third of the lock display. NOTE: Shim values should be adjusted slowly since there is usually a delay in the lock response. NOTE: When adjusting the onaxis (Z) shims the [ONAXIS] key must be activated (a lit LED on the button). Note also that the [FINE] key must be activated. NOTE: The rate of the sweep can be adjusted. First, press the [2ND] key and then the [SWEEP AMPL.] key. Then adjust the value by turning the control knob. The recommended value for the sweep rate is 0.50.

11 10 4 Leaving the Spectrometer Before you end your NMR session, the instrument should be returned to a stand-by condition with the deuterium lock engaged and shims set for the standard sample. This is particularly important if a solvent other than CDCl 3 has been used. Note that if variable temperature experiments have been performed, the probe must be returned to room temperature before these procedures are performed! Press [LIFT ON-OFF] to eject your sample Remove your sample and insert the solvent tube (CDCl 3 ) in the magnet Press [LIFT ON-OFF] to send the solvent tube down If a solvent other than CDCl 3 has been used, click on (lock). Then select CDCl3 from the menu and wait for the lock: finished message. Finally, enter rsh anshim *Optional* Adjust Z and Z 2 as described in step 3.5 *Optional* Press [AUTOSHIM] to activate the automatic shim routine. Note that before starting the autoshim routine, you should shim Z and Z 2 manually Close the acquisition window as follows: Left-click the red triangle on the upper right corner of the TopSpin window Click on to close the acquisition window Expand the Guest folder and drag EB into the data area Always sign the NMR logbook (your initials, time, experiments you have done, and solvent)! Shut (and lock) the door when you leave the room All unusual spectrometer behavior should be noted in the logbook and reported immediately to the NMR Facility Manager

12 1D EXPERIMENTS 11

13 H NMR Below is described the procedure to acquire a standard proton spectra. 5.1 Create an experiment Insert the sample (see step 3.3) (edc) Enter desired values for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_1H_routine Type the dataset title in the TITLE box If necessary, lock the spectrometer (see step 3.4) Optimize the Z and Z 2 (and probably X and Y) shims (see step 3.5) NOTE: Locking and shimming should be carried out at the beginning of every NMR session. However, it is not necessary to re-lock and re-shim if the sample is not changed between the experiments! 5.2 Acquisition and processing. Clicking this button sets receiver gain (rga), starts the acquisition (zg), and performs Fourier transform (ef), phase correction (apk) and baseline correction (abs). The whole process takes about 1 min 15 sec NOTE: If the automatic phase correction does not give satisfactory results, phase correction should be performed manually (see Appendix B). *If you are performing processing on your own PC, you can skip steps * 5.3 Calibration Expand the reference peak Move the red cursor line at the reference peak and left-click Enter the appropriate chemical shift value in ppm (i.e. for TMS 0) 5.4 Peak picking Define peak picking range button is automatically activated (green). The spectrum can be expanded when this button is inactive (gray) Put the cursor at the upper-left corner of a peak picking range

14 13 Left-click-hold and drag the mouse to the lower right corner of the range. The peak picking range will be marked green and the peaks in the range are picked and displayed Repeat the above two steps for each peak picking range to be defined to save peak picking and return, or click on to return without save NOTE: To define peaks manually: click on in the peak picking toolbar (button turns green). Put the red cursor line at the desired peak and left-click. To change peak picking ranges: click on (button turns green). Put the cursor on one of the edges of the peak picking range and left-click-hold and drag it to its new position. To delete all peaks: click on. To delete a specific peak: right-click on a defined peak and select Delete Peak Under Cursor. To delete all peak picking ranges: click on. To delete a specific range: right-click on a defined range and select Delete Region Under Cursor. NOTE: Peak picking can also be performed automatically using the command ppf. 5.5 Integration, then click on, and finally click on to delete the automatically determined integral regions, the button turns green Put the red cursor line at one edge of a peak or multiplet Left-click-hold and drag the cursor line to the other edge of the peak or multiplet Perform the above two steps for all peaks and/or multiplets to be integrated Right-click in the reference integral region Calibrate Enter the desired value for the reference integral to save integrals and return, or click on to return without save NOTE: The spectrum can be expanded when the define integral region button inactive (gray). is NOTE: A properly defined integral should extend beyond the apparent ends of the peak (if there is no other adjacent peak). Integral should also be properly phased. A properly phased integral should be horizontal before and after the peak. Do phasing as follows: right-click in the integral region to be phased and click on Select / Deselect (selected integral regions are indicated by a color filled integral region). If no integral is selected, phasing will work on all integrals. Left-click-hold (bias) or (slope) and move the mouse until the integral is phased.

15 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_1H+pp+int.xwp (peak and integral labels), +/AN_1H+pp.xwp (only peak labels) or +/AN_1H.xwp (no peak or integral labels) Select Use plot limits from screen / CY NOTE: TopSpin offers a multiplet analysis package. Enter mana to open the Multiplet Analysis mode. See Help/User s Guide for instructions.

16 C NMR Below is described the procedure to acquire a standard 1 H decoupled carbon spectra. 6.1 Create an experiment Insert the sample (see step 3.3) (edc) Enter desired values for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_13C Type the dataset title in the TITLE box If necessary, lock the spectrometer (see step 3.4) Optimize the Z and Z 2 (and probably X and Y) shims (see step 3.5) NOTE: Locking and shimming should be carried out at the beginning of every NMR session. However, it is not necessary to re-lock and re-shim if the sample is not changed between the experiments! 6.2 Acquisition and processing. Clicking this button sets receiver gain (rga), starts the acquisition (zg), and performs Fourier transform (ef), phase correction (apk) and baseline correction (abs). The whole process takes about 13 min NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value (this can be any number greater than 1). Check the acquisition time by clicking on. NOTE: It is possible to observe the real time Fourier transform while the acquisition is running. After a few scans have been acquired, enter tr, ef and apk. Click the red triangle on the upper right corner of the TopSpin window. Click on, the button turns green. Click on and select Window function: em, Phase correction mode: pk, and Baseline correction mode: quad. Click on. If you want to observe the FID, click on. NOTE: The acquisition can be stopped by clicking on (halt). WARNING! Clicking on (stop) will trash and delete the on-going data immediately! If you halt the experiment, do ef, apk and abs. NOTE: If the automatic phase correction does not give satisfactory results, phase correction should be performed manually (see Appendix B).

17 16 *If you are performing processing on your own PC, you can skip steps * 6.3 Calibration Expand the reference peak Move the red cursor line at the reference peak and left-click Enter the appropriate chemical shift value in ppm (i.e. for middle peak of CDCl 3 77) 6.4 Peak picking Define peak picking range button is automatically activated (green). The spectrum can be expanded when this button is inactive (gray) Put the cursor at the upper-left corner of a peak picking range Left-click-hold and drag the mouse to the lower right corner of the range. The peak picking range will be marked green and the peaks in the range are picked and displayed Repeat the above two steps for each peak picking range to be defined to save peak picking and return, or click on to return without save NOTE: To define peaks manually: click on in the peak picking toolbar (button turns green). Put the red cursor line at the desired peak and left-click. To change peak picking ranges: click on (button turns green). Put the cursor on one of the edges of the peak picking range and left-click-hold and drag it to its new position. To delete all peaks: click on. To delete a specific peak: right-click on a defined peak and select Delete Peak Under Cursor. To delete all peak picking ranges: click on. To delete a specific range: right-click on a defined range and select Delete Region Under Cursor. NOTE: Peak picking can also be performed automatically using the command ppf. 6.5 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_13C+pp.xwp (peak labels), +/AN_13C.xwp (no peak labels) Select Use plot limits from screen / CY

18 17 7 DEPT 45, DEPT 90, and DEPT 135 DEPT (Distortionless Enhancement by Polarization Transfer) is a polarization transfer technique used for the observation of nuclei with a small gyromagnetic ratio, which are J-coupled to 1 H (typically 13 C). DEPT 45 yields spectra with positive CH, CH 2, and CH 3 signals, DEPT 90 yields spectra with only CH signals, whereas DEPT 135 yields spectra with positive CH and CH 3 signals and negative CH 2 signals. 7.1 Acquire a standard 1D 13 C spectrum Acquire and process a standard 13 C spectrum as described in Chapter 6 (steps ) Expand the spectrum so that the 13 C signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 7.2 Create a DEPT experiment (edc) Enter a new value for EXPNO Click the down-arrow of the Experiment box and select AN_DEPT45, AN_DEPT90 or AN_DEPT135 Type the dataset title in the TITLE box 7.3 Set the acquisition and processing parameters Enter sw and enter the SW value recorded in step 7.1 Enter o1p and enter the O1P value recorded in step 7.1 Enter sr and enter the SR value recorded in step 7.1 NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value that is a multiple of 4. Check the acquisition time by clicking on. 7.4 Acquisition and Fourier transform Enter rga to automatically set the receiver gain Wait for the rga : finished message Enter zg to start the acquisition; the expected experiment time is ca. 13 min Ignore the Warning! by clicking Wait for the zg: finished message

19 18 Enter ef to perform Fourier transform with exponential function NOTE: It is possible to observe the real time Fourier transform while the acquisition is running. Click on, the button turns green. Click on and select Window function: em, Phase correction mode: pk, and Baseline correction mode: quad. Click on. If you want to observe the FID, click on. NOTE: The acquisition can be stopped by clicking on (halt). WARNING! Clicking on (stop) will trash and delete the on-going data immediately! 7.5 Phase and baseline correction Phase correction should be performed manually (see Appendix B) DEPT 45 and DEPT 90: all signals positive DEPT 135: CH and CH 3 positive, CH 2 negative. Before doing phase correction for DEPT 135, click on to shift the zero line of the spectrum to the center of the screen Enter abs to automatically correct the baseline *If you are performing processing on your own PC, you can skip step 7.6* 7.6 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_13C.xwp for DEPT 45 and DEPT 90, and +/AN_DEPT135.xwp for DEPT 135 Select Use plot limits from screen / CY It is helpful to display a normal 13 C spectrum along with the DEPT spectrum. In order to do this: Add a dataset as follows: left-click-hold the dataset in the browser and drag it into the data window NOTE: Click on to toggle between superimposed and stacked display. In order to shift and scale individual spectra: select one of the spectra by clicking it in the lower part of the browser. Left-click-hold and move the mouse to align the intensities. Left-click-hold and move the mouse to shift the spectrum baseline up or down. Click on to return from multiple display mode. NOTE: In order to plot multiple spectrum display, click on and select Print active window. Alternatively, use the TopSpin Plot Editor (see the TopSpin Plot Editor manual for instructions).

20 H, 1 H Correlation: Selective 1D COSY The selective 1D COSY (COrrelation SpectroscopY) experiment is the advanced 1D variant of the most common 2D experiment, 2D COSY (Chapter 16). Instead of recording the full 2D matrix, it is possible to measure one row by replacing the first 90 pulse of the COSY experiment with a selective excitation sequence, thus looking only for spin couplings that affect the particular proton excited. 8.1 Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ). However, before step 5.2, press [SPIN ON-OFF] to stop spinning Expand the peak to be irradiated to determine the correct O1 value for the COSY experiment Move the red cursor line on top of the peak to be irradiated and left-click Write down the value Repeat the above five steps for all peaks to be irradiated Enter sr and write down the value 8.2 Create a 1D COSY experiment (edc) Enter a new value for EXPNO Click the down-arrow of the Experiment box and select AN_1D_COSY Type the dataset title in the TITLE box 8.3 Set the acquisition and processing parameters Enter o1 and enter one of the O1 values recorded in step 8.1 The center of the spectrum is now the same as the chosen peak. Check that the SW value is large enough to include the whole spectrum. Change it if necessary: enter sw and enter a new value Enter sr and enter the SR value from step 8.1 NOTE: The length of the selective pulse P12 affects its selectivity. The achieved selectivity is approximately the reciprocal of the pulse length. In the parameter set AN_1D_COSY the value of P12 is set to 80 ms, corresponding to an excitation width of approximately 12.5 Hz. It is possible to change the length of the pulse P12 based on the selectivity desired. In such case, the pulse amplitude (i.e. the power level SP2) must also be adjusted to give a desired flip angle. Please see Chapter 12 for details on how to do this. The following predefined values can be used for P12 and SP2: p12 = 80 ms width 12.5 Hz sp2= 65 db p12= 55 ms width 18.2 Hz sp2= 61.9 db p12= 40 ms width 25.0 Hz sp2= 59 db

21 20 p12= 30 ms width 33.3 Hz sp2= 56.4 db NOTE: The delay D2 (1/2J(H,H)) determines the intensity of the signals that are spinspin coupled to the irradiated signal. It may be necessary to perform the experiment twice, for example in order to identify coupling partners with both small and large spinspin coupling constants. In the parameter set AN_1D_COSY the value of D2 is set to 50 ms, corresponding to a coupling constant of 10 Hz. In order to set the length of the delay, enter d2 and then enter a value between 30 and 60 ms. For multispin systems the delay D2 cannot always be optimized. In that case, an alternative is the selective 1D TOSCY experiment using a short spinlock (see Chapter 9). NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value that is a multiple of 8. Check the acquisition time by clicking on. NOTE: The spectral width (the SW value) can be reduced by using spoffs. See Appendix B for instructions. 8.4 Acquisition and Fourier transform Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 4 min Ignore the Warning! by clicking Wait for the zg: finished message Enter ef to perform Fourier transform with exponential function Repeat steps for each peak to be irradiated NOTE: If several peaks are to be irradiated, then the command multizg can be useful (see Appendix B). 8.5 Phase and baseline correction to shift the zero line of the spectrum to the center of the screen Phase correct each multiplet individually using manual phase correction (see Appendix B). Note that the irradiated signals are unperturbed and that the signals of the coupling partners show the active coupling in antiphase Enter abs to automatically correct the baseline *If you are performing processing on your own PC, you can skip step 8.6* 8.6 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_1H.xwp

22 21 Select Use plot limits from screen / CY It is helpful to display a normal 1 H spectrum along with the 1D COSY spectrum. In order to do this: Add a dataset as follows: left-click-hold the dataset in the browser and drag it into the data window NOTE: Click on to toggle between superimposed and stacked display. In order to shift and scale individual spectra: select one of the spectra by clicking it in the lower part of the browser. Left-click-hold and move the mouse to align the intensities. Left-click-hold and move the mouse to shift the spectrum baseline up or down. Click on to return from multiple display mode. NOTE: In order to plot multiple spectrum display, click on and select Print active window. Alternatively, use the TopSpin Plot Editor (see the TopSpin Plot Editor manual for instructions).

23 22 9 Extended 1 H, 1 H Correlation: Selective 1D TOCSY The selective 1D TOCSY (TOtal Correlation SpectroscopY) experiment is the advanced 1D variant of the 2D TOCSY experiment described in Chapter 18. Instead of recording the full 2D matrix, it is possible to measure one row by selective excitation, thus looking only for spin couplings that affect the particular proton excited. 9.1 Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ). However, before step 5.2, press [SPIN ON-OFF] to stop spinning Expand the peak to be irradiated to determine the correct O1 value for the TOCSY experiment Move the red cursor line on top of the peak to be irradiated and left-click Write down the value Repeat the above five steps for all peaks to be irradiated Enter sr and write down the value 9.2 Create a 1D TOCSY experiment (edc) Enter a new value for EXPNO Click the down-arrow of the Experiment box and select AN_1D_TOCSY Type the dataset title in the TITLE box 9.3 Set the acquisition and processing parameters Enter o1 and enter one of the O1 values recorded in step 9.1 The center of the spectrum is now the same as the chosen peak. Check that the SW value is large enough to include the whole spectrum. Change it if necessary: enter sw and enter a new value Enter sr and enter the SR value from step 9.1 NOTE: The length of the selective pulse P12 affects its selectivity. The achieved selectivity is approximately the reciprocal of the pulse length. In the parameter set AN_1D_TOCSY the value of P12 is set to 80 ms, corresponding to an excitation width of approximately 12.5 Hz. It is possible to change the length of the pulse P12 based on the selectivity desired. In such case, the pulse amplitude (i.e. the power level SP2) must also be adjusted to give a desired flip angle. Please see Chapter 12 for details on how to do this. The following predefined values can be used for P12 and SP2: p12 = 80 ms width 12.5 Hz sp2= 65 db p12 = 55 ms width 18.2 Hz sp2= 61.9 db p12 = 40 ms width 25.0 Hz sp2= 59 db p12 = 30 ms width 33.3 Hz sp2= 56.4 db

24 23 NOTE: In the 1D TOCSY experiment, the parameter D9 determines the length of the TOCSY mixing time. This parameter determines how far the spin coupling network will be probed. In the parameter set AN_1D_TOCSY, the value of D9 is set to 80 ms. Short mixing times (20 40 ms) produce cross signals practically only between directly coupled nuclei (as in COSY). For long mixing times ( ms), correlation with more distant protons can be observed. In order to set the length of the spinlock period, enter d9 and then enter the appropriate value. NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value that is a multiple of 8. Check the acquisition time by clicking on. NOTE: The spectral width (the SW value) can be reduced by using spoffs. See Appendix B for instructions. 9.4 Acquisition and Fourier transform Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 14 min Ignore the Warning! by clicking Wait for the zg: finished message Enter ef to perform Fourier transform with exponential function Repeat steps for each peak to be irradiated NOTE: If several peaks are to be irradiated, then the command multizg can be useful (see Appendix B). 9.5 Phase and baseline correction Phase correct the spectrum manually (see Appendix B), so that the irradiated peak is phased up. The TOCSY peaks will also be positive Enter abs to automatically correct the baseline *If you are performing processing on your own PC, you can skip step 9.6* 9.6 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_1H.xwp Select Use plot limits from screen / CY It is helpful to display a normal 1 H spectrum along with the 1D TOCSY spectrum. In order to do this:

25 24 Add a dataset as follows: left-click-hold the dataset in the browser and drag it into the data window NOTE: Click on to toggle between superimposed and stacked display. In order to shift and scale individual spectra: select one of the spectra by clicking it in the lower part of the browser. Left-click-hold and move the mouse to align the intensities. Left-click-hold and move the mouse to shift the spectrum baseline up or down. Click on to return from multiple display mode. NOTE: In order to plot multiple spectrum display, click on and select Print active window. Alternatively, use the TopSpin Plot Editor (see the TopSpin Plot Editor manual for instructions).

26 H, 1 H Correlation Through Space: Selective 1D NOESY, ROESY, and off-res-roesy In the 1D NOESY/ROESY (Nuclear Overhauser Effect SpectroscopY/Rotating-frame Overhauser Effect SpectroscopY) experiment, selective irradiation of one group of protons causes a change in the intensities of other signals. This change is related to the inverse sixth power of the distance between the spins. The choice between the 1D NOESY and 1D ROESY experiments depends on the molecular tumbling rate, which is in large part determined by the molecular weight. The 1D off-resonance ROESY experiment can be considered a combination of the NOESY and ROESY experiments. It provides both efficient suppression of HOHAHA (HOmonuclear HArtmann-HAhn) transfer of magnetization and reduction of offset effects, which are problems associated with the standard ROESY experiment. Please read Appendix C before doing these experiments! 10.1 Tune and match the probe for 1 H observation If necessary, the probehead should be tuned and matched for 1 H observation. Follow the instructions given in Appendix D Tuning and matching 1 H Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ). However, before step 5.2, press [SPIN ON-OFF] to stop spinning Expand the peak to be irradiated to determine the correct O1 value for the NOESY/ROESY experiment Move the red cursor line on top of the peak to be irradiated and left-click Write down the value Repeat the above five steps for all peaks to be irradiated Enter sr and write down the value 10.3 Create a 1D NOESY/ROESY/off-res-ROESY experiment (edc) Enter a new value for EXPNO Click the down-arrow of the Experiment box and select AN_1D_NOESY, AN_1D_ROESY or AN_1D_off-res-ROESY Type the dataset title in the TITLE box

27 Set the acquisition and processing parameters Enter o1 and enter one of the O1 values recorded in step 10.2 The center of the spectrum is now the same as the chosen peak. Check that the SW value is large enough to include the whole spectrum. Change it if necessary: enter sw and enter a new value Enter sr and enter the SR value from step D off-res-roesy only: Click the AcquPars tab Click on Edit... next to FQLIST Click on next to FQ1LIST Select an existing list or create a new file Click on, a text editor opens First text row: o Second text row: the O1 value from step 10.2 Third text row: the O1 value from step Save and close the list Click on Click the Spectrum tab NOTE: The length of the selective pulse P12 affects its selectivity. The achieved selectivity is approximately the reciprocal of the pulse length. In the parameter sets AN_1D_NOESY/ROESY/off-res-ROESY the value of P12 is set to 80 ms, corresponding to an excitation width of approximately 12.5 Hz. It is possible to change the length of the pulse P12 based on the selectivity desired. In such case, the pulse amplitude (i.e. the power level SP2) must also be adjusted to give a desired flip angle. Please see Chapter 12 for details on how to do this. The following predefined values can be used for P12 and SP2: p12 = 80 ms width 12.5 Hz sp2= 65 db p12 = 55 ms width 18.2 Hz sp2= 61.9 db p12 = 40 ms width 25.0 Hz sp2= 59 db p12 = 30 ms width 33.3 Hz sp2= 56.4 db NOTE: In the 1D NOESY, the parameter D8 determines the length of the mixing period during which NOE buildup occurs. This should be on the order of T 1 relaxation time. In the parameter set AN_1D_NOESY, the value of D8 is set to 500 ms. This value can be quickly optimized using the procedure described in step In order to set the length of the mixing period, enter d8 and then enter the appropriate value. NOTE: In the 1D ROESY/off-res-ROESY, the pulse P15 sets the length of the cw spinlock pulse. In this parameter set, the value of P15 is set to 500 ms. A good rule of thumb is that P15 for the ROESY experiment of a molecule should be the same as D8 for the NOESY experiment of that molecule. In order to set the length of the spinlock pulse, enter p15 and then enter the appropriate value.

28 27 NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value that is a multiple of 2. Check the acquisition time by clicking on. NOTE: The spectral width (the SW value) can be reduced by using spoffs. See Appendix B for instructions Acquisition and Fourier transform Enter rga to automatically set the receiver gain Wait for the rga: finished message. 1D NOESY only: enter rg and increase the value by 100%; click on Enter zg to start the acquisition; the expected experiment time is ca. 15 min Ignore the Warning! by clicking Wait for the zg: finished message Enter ef to perform Fourier transform with exponential function Repeat steps for each peak to be irradiated NOTE: If several peaks are to be irradiated, then the command multizg can be useful (see Appendix B) Phase and baseline correction to shift the zero line of the spectrum to the center of the screen Phase correct the spectrum manually (see Appendix B), so that the large, irradiated peak is phased down. The NOE peaks will be positive for small molecules (MW < 600) and negative for large molecules (MW > 1500). The ROE peaks are always positive. Artifacts are sometimes present. These will be anti-phase or dispersive and occur only for spins J coupled to the selected peak Enter abs to automatically correct the baseline NOTE: The intensity of the NOE/ROE peaks is usually very small compared to that of the irradiated peak. Therefore, left-click-hold and move the mouse to change the intensity of the NOE/ROE peaks. *If you are performing processing on your own PC, you can skip step 10.7* 10.7 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_1D_NOE(ROE).xwp Select Use plot limits from screen / CY

29 28 It is helpful to display a normal 1 H spectrum along with the 1D NOESY/ROESY/offres-ROESY spectrum. In order to do this: Add a dataset as follows: left-click-hold the dataset in the browser and drag it into the data window NOTE: Click on to toggle between superimposed and stacked display. In order to shift and scale individual spectra: select one of the spectra by clicking it in the lower part of the browser. Left-click-hold and move the mouse to align the intensities. Left-click-hold and move the mouse to shift the spectrum baseline up or down. Click on to return from multiple display mode. NOTE: In order to plot multiple spectrum display, click on and select Print active window. Alternatively, use the TopSpin Plot Editor (see the TopSpin Plot Editor manual for instructions).

30 29 11 Highly Selective Pseudo 2D Experiments: CSSF- TOCSY, CSSF-NOESY, and CSSF-off-res- ROESY The chemical shift selective filtration (CSSF) is a powerful method for separation of severely overlapping proton signals. The CSSF experiments enable separate selective NMR spectra to be acquired for overlapped signals with chemical shift differences as small as a few Hertz. However, sometimes small peaks arising from a peak that is overlapped with the selected peak may appear in the spectra. In order to run a successful CSSF experiment, two requirements must be fulfilled. First, the selected peak must have a distinct chemical shift, as the filter will not be able to distinguish between two signals with identical chemical shifts. Second, the precise chemical shift of the selected peak must be known. See P. T. Robinson et al., J. Magn. Reson., 170 (2004) 97 and S. J. Duncan et al., Magn. Reson. Chem., 45 (2007) 283 for more information on CSSF experiments Tune and match the probe for 1 H observation If necessary, the probehead should be tuned and matched for 1 H observation. Follow the instructions given in Appendix D Tuning and matching 1 H Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ). However, before step 5.2, press [SPIN ON-OFF] to stop spinning Expand the peak to be irradiated to determine the correct O1 value for the CSSF experiment Move the red cursor line on top of the peak to be irradiated and left-click Write down the value Enter sr and write down the value NOTE: the exact chemical shift (the O1 value) must be identified from the 1 H spectrum. If this is not possible, the individual chemical shift must be determined from another experiment, such as a selective 1D experiment, or a normal 2D experiment Create a CSSF experiment (edc) Enter a new value for EXPNO Click the down-arrow of the Experiment box and select AN_CSSF-TOCSY, AN_CSSF-NOESY or AN_CSSF-off-res-ROESY Type the dataset title in the TITLE box

31 Set the acquisition and processing parameters Click the AcquPars tab Enter the O1 value from step 11.2 Enter the SW value for the F2 Frequency axis. The center of the spectrum is the same as the chosen peak. Check that the SW value is large enough to include the whole spectrum *Optional* Set the parameter TD for the F1 Frequency axis as described in the NOTE below *Optional* Click on and set the parameters D21 and IN21 as described in the NOTE below Click the ProcPars tab Enter the SR value from step 11.2 for the F2 Frequency axis Click the Spectrum tab NOTE: The optimal value for the length of the CSSF, t max, can be calculated from equation t max = 0.5/Δν, where Δν is the chemical shift difference between the overlapping signals. Usually, t max is set longer than the calculated value to account for the effects of relaxation. The length of t max can be adjusted by adjusting the number of CSSF increments. This can be done by changing the value of TD for the F1 frequency axis (the default value is 8). The two other parameters that influence on t max are the increment delay D21 (default 1 ms) and the increment for the latter, IN21 (default 10 ms). NOTE: All the other acquisition and processing parameters should be adjusted as described in the section Set the acquisition and processing parameters in Chapter 9 or Acquire and process a CSSF spectrum Enter rga to automatically set the receiver gain Wait for the rga: finished message. CSSF-NOESY only: enter rg and increase the value by 100%; click on Enter zg to start the acquisition Ignore the Warning! by clicking Wait for the zg: finished message Enter xf2 to perform Fourier transform in the F2 dimension Select Processing/Calculate Projections Select Calculate Sum Change the following parameters: Projection (sum) of rows Display projection as 1D First row/col 1 Last row/col the value of TD for the F1 Frequency axis (default 8) Destination PROCNO enter desired value for the processing number

32 31. The extracted row is stored as a 1D dataset under the specified PROCNO and displayed in a new data window 11.6 Phase and baseline correction Perform phase and baseline correction as described in the section Phase and baseline correction in Chapter 9 or Printing Print the spectrum as described in the section Printing in Chapter 9 or 10.

33 32 12 Calibration of the DPFGSE Sequence Soft pulses selectively excite only one multiplet of a 1 H NMR spectrum. Important characteristics of a soft pulse include the length, the amplitude, and the shape. The selectivity of a pulse is measured by its ability to excite a certain resonance (or group of resonances) without affecting near neighbors. Since the length of the selective pulse affects its selectivity, the length is selected based on the selectivity desired and then the pulse amplitude (i.e. power level) is adjusted to give a desired flip angle. The achieved selectivity is approximately the reciprocal of the pulse length. For example, if a multiplet of 40 Hz width is to be excited, then the duration of the applied selective pulse must be of the order of 25 ms. The selective 1D experiments and the highly selective pseudo 2D experiments employ the DPFGSE (Double Pulsed Field Gradient Spin Echo COrrelation SpectroscopY) sequence. The DPFGSE sequence itself can be used to pulse calibration and assessment of degree of selectivity (K. Stott et al., J. Magn. Reson., 125 (1997) 302). In this experiment, a nonselective 90 pulse is followed by the DPFGSE sequence. The selective pulses are set on resonance with the desired signal. This sequence will give the maximum signal when selective pulses are closest to inversion pulses. Therefore, pulse calibration can easily be achieved by varying the power level (or other parameter that describe the selective pulses) in order to give the maximum signal Tune and match the probe for 1 H observation If necessary, the probehead should be tuned and matched for 1 H observation. Follow the instructions given in Appendix D Tuning and matching 1 H Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ). However, before step 5.2, press [SPIN ON-OFF] to stop spinning Expand the peak to be irradiated to determine the correct O1 value for the DPFGSE experiment Move the red cursor line on top of the peak to be irradiated and left-click Write down the value 12.3 Create a DPFGSE experiment (edc) Enter a new value for EXPNO Click the down-arrow of the Experiment box and select AN_DPFGSE Type the dataset title in the TITLE box

34 Set the acquisition parameters Enter o1 and enter the O1 value recorded in step 12.2 The center of the spectrum is now the same as the chosen peak. Check that the SW value is large enough to include the whole spectrum. Change it if necessary: enter sw and enter a new value Enter p12 and enter the length of the selective pulse (based on the selectivity desired). Some examples: p12 = 80 ms width 12.5 Hz p12 = 55 ms width 18.2 Hz p12 = 40 ms width 25.0 Hz p12 = 30 ms width 33.3 Hz Note that the length of the selective pulse is in the order of milliseconds (ms). For example, to set the value of P12 to 55 ms, you must enter 55m Enter sp2 and enter an appropriate value for the power level of the selective pulse (the default unit is db). Some examples: p12 = 80 ms sp2 69 db p12 = 55 ms sp2 66 db p12 = 40 ms sp2 63 db p12 = 30 ms sp2 61 db This value will be optimized later (in step 12.6) 12.5 Acquire and process a DPFGSE spectrum Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 15 sec Ignore the Warning! by clicking Wait for the zg: finished message Enter ef to perform Fourier transform with exponential function. The irradiated resonance should appear in the middle of the window and no other peaks should be visible Phase correct the spectrum manually (see Appendix B) so that the irradiated peak is positive Reduce the spectral width by entering swh and changing the value to 1000 Hz Do zg, ef and phase correction once again Expand the spectrum so that the irradiated peak occupies approximately the center quarter of the window Enter dpl to save the displayed region 12.6 Perform the pulse calibration Enter paropt and answer the questions as follows: Enter parameter to modify sp2 Enter initial parameter value enter an appropriate value depending on the value of P12 (see step 12.4)

35 34 Enter parameter increment -2 Enter # of experiments 10 At the end of the experiment, the message paropt finished is displayed. Write down the value of SP2 and click on. This is the approximate power level for a pulse time selected in step 12.4 to view the spectra To obtain a more accurate value for SP2, parameter optimization must be repeated using a smaller parameter increment: Enter rep 1 to return to the original DPFGSE spectrum Enter paropt and answer the questions as follows: Enter parameter to modify sp2 Enter initial parameter value a value slightly above the power level determined above Enter parameter increment a value between -1 and -0.1 Enter # of experiments a value between 5 and 10 Write down the value of SP2 and click on. This is the accurate power level for the selective pulse P12 to view the spectra NOTE: This procedure can be repeated as many times as necessary using increasingly smaller parameter increment. Note that the smallest useful increment is -0.1.

36 35 13 Direct 1 H, 13 C Correlation: SELINCOR SELINCOR (SELective INverse H,C CORrelation) yields 1D 1 H spectra in which the desired proton signal is selected via a selective pulse on the directly bonded 13 C nucleus using the 1 J CH spin-spin coupling. In a SELINCOR spectrum, only protons directly attached to a specific carbon will be present Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ). However, before step 5.2, press [SPIN ON-OFF] to stop spinning Enter sr and write down the value 13.2 Acquire a standard 1D 13 C spectrum Acquire and process a standard 13 C spectrum as described in Chapter 6 (steps ) Expand the peak to be irradiated to determine the correct O2 value for the SELINCOR experiment Move the red cursor line on top of the peak to be irradiated and left-click Write down the value 13.3 Create a SELINCOR experiment (edc) Enter desired value for EXPNO Click the down-arrow of the Experiment box and select AN_SELINCOR Type the dataset title in the TITLE box 13.4 Set the acquisition and processing parameters Enter o2 and enter one of the O2 values recorded in step 13.2 Enter sr and enter the SR value from step 13.1 NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value (this can be any number greater than 1). Check the acquisition time by clicking on Acquisition and Fourier transform Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 22 min

37 36 Ignore the Warning! by clicking Wait for the zg: finished message Enter ef to perform Fourier transform with exponential function Repeat steps for each peak to be irradiated NOTE: If several peaks are to be irradiated, then the command multizg can be useful (see Appendix B) Phase and baseline correction Enter apk to automatically phase correct the spectrum Enter abs to automatically correct the baseline NOTE: If the automatic phase correction does not give satisfactory results, phase correction should be performed manually (see Appendix B). *If you are performing processing on your own PC, you can skip step 13.7* 13.7 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_1H.xwp Select Use plot limits from screen / CY It is helpful to display a normal 1 H spectrum along with the SELINCOR spectrum. In order to do this: Add a dataset as follows: left-click-hold the dataset in the browser and drag it into the data window NOTE: Click on to toggle between superimposed and stacked display. In order to shift and scale individual spectra: select one of the spectra by clicking it in the lower part of the browser. Left-click-hold and move the mouse to align the intensities. Left-click-hold and move the mouse to shift the spectrum baseline up or down. Click on to return from multiple display mode. NOTE: In order to plot multiple spectrum display, click on and select Print active window. Alternatively, use the TopSpin Plot Editor (see the TopSpin Plot Editor manual for instructions).

38 F NMR Below is described the procedure to acquire a standard 1 H coupled and decoupled 19 F spectra Create an experiment Insert the sample (see step 3.3) (edc) Enter desired values for NAME and EXPNO (PROCNO = 1, DIR = D:\Bruker\TOPSPIN, USER = your user name) Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_19F ( 1 H coupled) or AN_19F_dec ( 1 H decoupled) Type the dataset title in the TITLE box Enter ii to initialize the interface Wait for the ii: finished message; you will hear a snap from the probe as the pneumatic switch goes to the position 3 ( 19 F) Check that the green 19F LED is lit on the front face of the QNP Control Unit (at the bottom of the HPPR unit that is the module on the floor next to the magnet) If necessary, lock the spectrometer (see step 3.4) Tune and match the probehead. Follow the instructions given in Appendix D Tuning and matching 19 F/ 31 P Optimize the Z and Z 2 (and probably X and Y) shims (see step 3.5) NOTE: Locking and shimming should be carried out at the beginning of every NMR session. However, it is not necessary to re-lock and re-shim if the sample is not changed between the experiments! 14.2 Acquisition and processing. Clicking this button sets receiver gain (rga), starts the acquisition (zg), and performs Fourier transform (ef), phase correction (apk) and baseline correction (abs). The whole process takes about 1 min 15 sec NOTE: If the automatic phase correction does not give satisfactory results, phase correction should be performed manually (see Appendix B). *If you are performing processing on your own PC, you can skip steps *

39 Calibration Expand the reference peak Move the red cursor line at the reference peak and left-click Enter the appropriate chemical shift value in ppm 14.4 Peak picking Define peak picking range button is automatically activated (green). The spectrum can be expanded when this button is inactive (gray) Put the cursor at the upper-left corner of a peak picking range Left-click-hold and drag the mouse to the lower right corner of the range. The peak picking range will be marked green and the peaks in the range are picked and displayed Repeat the above two steps for each peak picking range to be defined to save peak picking and return, or click on to return without save NOTE: To define peaks manually: click on in the peak picking toolbar (button turns green). Put the red cursor line at the desired peak and left-click. To change peak picking ranges: click on (button turns green). Put the cursor on one of the edges of the peak picking range and left-click-hold and drag it to its new position. To delete all peaks: click on. To delete a specific peak: right-click on a defined peak and select Delete Peak Under Cursor. To delete all peak picking ranges: click on. To delete a specific range: right-click on a defined range and select Delete Region Under Cursor. NOTE: Peak picking can also be performed automatically using the command ppf. *****If the coupled experiment was performed, the 19 F spectrum can be integrated as described in step 5.5***** 14.5 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_19F_31P+pp+int.xwp (peak and integral labels), +/AN_19F_31P+pp.xwp (only peak labels) or +/AN_19F_31P.xwp (no peak or integral labels) Select Use plot limits from screen / CY

40 Return the QNP switch back to the position 1 ( 13 C) Open a dataset that contain 1 H acquisition parameters. OR: Click on, change the EXPNO, select AN_1H_routine, and click on Enter ii to initialize the interface Wait for the ii: finished message; the pneumatic switch goes to the position 1 ( 13 C) Check that the green 13C LED is lit on the front face of the QNP Control Unit Tune and match 1 H and 13 C (see Appendix D)

41 P NMR Below is described the procedure to acquire a standard 1 H coupled and decoupled 31 P spectra Create an experiment Insert the sample (see step 3.3) (edc) Enter desired values for NAME and EXPNO (PROCNO = 1, DIR = D:\Bruker\TOPSPIN, USER = your user name) Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_31P ( 1 H coupled) or AN_31P_dec ( 1 H decoupled) Type the dataset title in the TITLE box Enter ii to initialize the interface Wait for the ii: finished message; you will hear a snap from the probe as the pneumatic switch goes to the position 2 ( 31 P) Check that the green 31P LED is lit on the front face of the QNP Control Unit (at the bottom of the HPPR unit that is the module on the floor next to the magnet) If necessary, lock the spectrometer (see step 3.4) Tune and match the probehead. Follow the instructions given in Appendix D Tuning and matching 19 F/ 31 P Optimize the Z and Z 2 (and probably X and Y) shims (see step 3.5) NOTE: Locking and shimming should be carried out at the beginning of every NMR session. However, it is not necessary to re-lock and re-shim if the sample is not changed between the experiments! 15.2 Acquisition and processing. Clicking this button sets receiver gain (rga), starts the acquisition (zg), and performs Fourier transform (ef), phase correction (apk) and baseline correction (abs). The whole process takes about 1 min 30 sec NOTE: If the automatic phase correction does not give satisfactory results, phase correction should be performed manually (see Appendix B). *If you are performing processing on your own PC, you can skip steps *

42 Calibration Expand the reference peak Move the red cursor line at the reference peak and left-click Enter the appropriate chemical shift value in ppm 15.4 Peak picking Define peak picking range button is automatically activated (green). The spectrum can be expanded when this button is inactive (gray) Put the cursor at the upper-left corner of a peak picking range Left-click-hold and drag the mouse to the lower right corner of the range. The peak picking range will be marked green and the peaks in the range are picked and displayed Repeat the above two steps for each peak picking range to be defined to save peak picking and return, or click on to return without save NOTE: To define peaks manually: click on in the peak picking toolbar (button turns green). Put the red cursor line at the desired peak and left-click. To change peak picking ranges: click on (button turns green). Put the cursor on one of the edges of the peak picking range and left-click-hold and drag it to its new position. To delete all peaks: click on. To delete a specific peak: right-click on a defined peak and select Delete Peak Under Cursor. To delete all peak picking ranges: click on. To delete a specific range: right-click on a defined range and select Delete Region Under Cursor. NOTE: Peak picking can also be performed automatically using the command ppf. *****If the coupled experiment was performed, the 31 P spectrum can be integrated as described in step 5.5***** 15.5 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_19F_31P+pp+int.xwp (peak and integral labels), +/AN_19F_31P+pp.xwp (only peak labels) or +/AN_19F_31P.xwp (no peak or integral labels) Select Use plot limits from screen / CY

43 Return the QNP switch back to the position 1 ( 13 C) Open a dataset that contain 1 H acquisition parameters. OR: Click on, change the EXPNO, select AN_1H_routine, and click on Enter ii to initialize the interface Wait for the ii: finished message; the pneumatic switch goes to the position 1 ( 13 C) Check that the green 13C LED is lit on the front face of the QNP Control Unit Tune and match 1 H and 13 C (see Appendix D)

44 2D EXPERIMENTS 43

45 H, 1 H Correlation: COSY COSY (COrrelation SpectroscopY) is a homonuclear 2D technique that is used to correlate the chemical shifts of 1 H nuclei, which are J-coupled to one another. The COSY spectrum is displayed in magnitude mode. Consequently, a typical spectral resolution is sufficient only for resolving large scalar couplings. If a higher resolution is desired then the DQF-COSY experiment should be performed (see Chapter 17) Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 16.2 Create a COSY experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_COSY Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 16.2, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 16.1 for both the F2 and F1 Frequency axis Enter the O1P value from step 16.1 Click the ProcPars tab Enter the SR value from step 16.1 for both the F2 and F1 Frequency axis Click the Spectrum tab 16.4 Acquisition, Fourier transform and baseline correction Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 17 min

46 45 Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation Enter absb to automatically correct the baseline NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 16.1 EXPNO the experiment number of the sample measured in step 16.1 PROCNO the processing number of the sample measured in step 16.1 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 and F1 frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined diagonal peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step Printing Define the print region using the mouse Select Print with layout plot directly

47 46 Select LAYOUT= +/AN_2D_1H-1H.xwp Select Use plot limits from screen / CY Select Fill data set list with projections NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

48 H, 1 H Correlation: DQF-COSY The DQF-COSY (DQF = Double Quantum Filtered) experiment is a slightly modified COSY, which allows for the observation of double quantum coherence. One advantage of the DQF-COSY experiment is the phase sensitivity. In general, phase sensitive spectrum has a higher resolution than an otherwise equivalent magnitude spectrum (COSY). Another advantage is the partial cancellation of the diagonal peaks in a DQF- COSY spectrum. This makes cross peaks near the diagonal easier to distinguish. The DQF-filter also has the advantage that the singlet peaks (including solvent peaks) are eliminated or reduced in intensity. The drawback is that due to the DQF-filter there is a sensitivity loss compared with the COSY experiment. The other disadvantage of the DQF-COSY experiment arises from the increased complexity of processing phase sensitive data Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 17.2 Create a DQF-COSY experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_DQF-COSY Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 17.2, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 17.1 for both the F2 and F1 Frequency axis Enter the O1P value from step 17.1 Click the ProcPars tab Enter the SR value from step 17.1 for both the F2 and F1 Frequency axis Click the Spectrum tab

49 Acquisition and Fourier transform Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter rg and reduce the value by 50%; click on Enter zg to start the acquisition; the expected experiment time is ca. 1 h Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 17.1 EXPNO the experiment number of the sample measured in step 17.1 PROCNO the processing number of the sample measured in step 17.1 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Phase and baseline correction Generally, the 2D spectrum is first phase corrected in the F2 dimension (rows), and then in the F1 dimension (columns). For the correction in the F2 dimension, three rows each with a cross peak should be selected (preferably on the same side of the diagonal). The cross peak of one row should be the far left of the spectrum, the cross peak of the second row should be close to the middle, and the one for the third row should be the far right of the spectrum. For the correction in the F1 dimension, three columns rather than rows should be selected. The phase correction of the DQF-COSY is best performed while examining the cross peaks rather than the diagonal peaks. When the spectrum is phased properly, the cross peaks will be purely absorptive and they will not have the slowly decaying wings typical to dispersion peaks. However, since in the DQF-COSY spectrum each multiplet

50 49 has adjacent positive and negative peaks (they are antiphase), it is not possible to phase the spectrum so that all peaks are positive. Expand a cross peak at the far-left-down of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a cross peak at the middle of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a cross peak at the far-right-up of the spectrum Right-click at the peak position and select Add to display the full spectrum to phase correct rows, or click on to phase correct columns By default, all rows/columns are selected as indicated by the filled blue squares. To select one row/column: left-click in the corresponding part of the data window. To select all the rows/columns: click on. The red vertical line indicates the default pivot point in the upper row/column. To change the pivot point: right-click at peak and select Set Pivot Point. Left-click-hold (zero order phase correction) and move the mouse until the reference peak of the first row/column is exactly in absorption mode Left-click-hold (first order phase correction) and move the mouse until the reference peak of the second and third row/column is exactly in absorption mode to save and return, or click on to return without save to return from 2D Phase Mode Enter absb to automatically correct the baseline 17.7 Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 and F1 frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined diagonal peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step Printing Define the print region using the mouse Select Print with layout plot directly

51 50 Select LAYOUT= +/AN_2D_1H-1H.xwp Select Use plot limits from screen / CY Select Fill data set list with projections NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

52 51 18 Extended 1 H, 1 H Correlation: TOCSY TOCSY (TOtal Correlation SpectroscopY) is a homonuclear 2D technique that do not simply display cross peaks between pairs of J-coupled (geminal or vicinal) protons, but also display cross peaks between pairs of protons that are not directly J-coupled but are in the same J-coupling network or spin system. This technique is therefore mostly used for peptides or oligosaccharides, since it serves the identification of isolated subunits Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 18.2 Create a TOCSY experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_TOCSY Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 18.2, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 18.1 for both the F2 and F1 Frequency axis Enter the O1P value from step 18.1 Click the ProcPars tab Enter the SR value from step 18.1 for both the F2 and F1 Frequency axis Click the Spectrum tab NOTE: In the TOCSY experiment, the parameter D9 determines the length of the TOCSY mixing time. This parameter determines how far the spin coupling network will be probed. In the parameter set AN_TOCSY, the value of D9 is set to 80 ms. Short mixing times (20 40 ms) produce cross signals practically only between directly coupled nuclei (as in COSY). For long mixing times ( ms), correlation with

53 52 more distant protons can be observed. In order to set the length of the spinlock period, enter d9 and then enter the appropriate value Acquisition and Fourier transform Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 1 h 25 min Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 18.1 EXPNO the experiment number of the sample measured in step 18.1 PROCNO the processing number of the sample measured in step 18.1 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Phase and baseline correction Correct the phase of the diagonal signals so that they are all positive. Then the true TOCSY cross peaks will all also be positive. To simplify the phasing of the 2D TOCSY spectrum, it helps to phase correct the second row: Enter rser 1 to transfer the first row to the 1D data set ~TEMP/1/1 Enter sinm to apply the sine-bell windowing function Enter ft to perform Fourier transform Manually phase correct the signals so that they are all positive (see Appendix B) to save the phase correction to the source 2D dataset

54 53 to return from 1D Phase Mode to return the 2D data set Enter xfb to Fourier transform the TOCSY spectrum again, this time applying the appropriate phase correction to F2 dimension The spectrum should now require additional phase correction only in F1 dimension: Expand a diagonal peak at the far left of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a diagonal peak at the middle of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a diagonal peak at the far right of the spectrum Right-click at the peak position and select Add to display the full spectrum to phase correct columns By default, all columns are selected as indicated by the filled blue squares. To select one column: left-click in the corresponding part of the data window. To select all the columns: click on. The red vertical line indicates the default pivot point in the upper column. To change the pivot point: right-click at peak and select Set Pivot Point. Left-click-hold (zero order phase correction) and move the mouse until the phase of the reference peak of the first column is positive Left-click-hold (first order phase correction) and move the mouse until the phase of the reference peak of the second and third column is positive to save and return, or click on to return without save to return from 2D Phase Mode Enter absb to automatically correct the baseline 18.7 Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 and F1 frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined diagonal peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step 18.1.

55 Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-1H.xwp Select Use plot limits from screen / CY Select Fill data set list with projections NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

56 H, 1 H Correlation Through Space: NOESY and gs-noesy 2D NOESY (Nuclear Overhauser Effect SpectroscopY) is a homonuclear experiment that yields correlation signals, which are caused by dipolar cross-relaxation between nuclei (usually protons) in a close spatial relationship. There are two different parameter sets available for NOESY: AN_NOESY for the standard NOESY experiment and AN_NOESYgs for the gradient selected version of NOESY. Of these two experiments, the standard NOESY is the better choice because this experiment employs the ZQF (Zero-Quantum Filter) sequence to effectively suppress the anti-phase dispersive components arising from the zero-quantum coherence. However, the gs-noesy experiment can be performed in a fraction of time compared with the standard NOESY experiment. Both experiments give exactly the same data. Please read Appendix C before doing these experiments! 19.1 Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 19.2 Create a NOESY experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_NOESY (recommendable) or AN_NOESYgs Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 19.2, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 19.1 for both the F2 and F1 Frequency axis Enter the O1P value from step 19.1 Click the ProcPars tab Enter the SR value from step 19.1 for both the F2 and F1 Frequency axis

57 56 Click the Spectrum tab 19.4 Optimize mixing time *****Step 19.4 is optional***** The parameter D8 determines the length of the mixing period during which NOE buildup occurs. This should be on the order of T 1 relaxation time. In the parameter sets AN_NOESY and AN_NOESYgs the value of D8 is set to 500 ms. However, you can optimize this value by yourself using quick and easy procedure described below. (edc) Change the EXPNO to create a 1D dataset from the 2D NOESY dataset Click the AcquPars tab. Select Change dimension from 2D to 1D and click on next to PULPROG and select zg Set NS to 1 and DS to 0 Click the Spectrum tab Enter zg. When the acquisition has finished enter ef Manually phase correct the spectrum (see Appendix B) Click the AcquPars tab next to PULPROG and select t1ir1d (inversion recovery sequence) Click the Spectrum tab Enter d7 1m to set the delay for inversion recovery to 1 msec Enter zg and efp to record and process a spectrum; the signals should all be negative Enter d7 2 to set the delay to 2 sec and then record and process another spectrum using zg and efp; the signals should all be positive Find a value for D7 in the range of 100 msec 2 sec, where all the signals are minimal Return to the 2D NOESY dataset Enter d8 and enter the value of D7 determined above 19.5 Acquisition and Fourier transform Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 1 h 40 min for the standard NOESY and ca. 50 min for the gs-noesy Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation

58 57 NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 19.1 EXPNO the experiment number of the sample measured in step 19.1 PROCNO the processing number of the sample measured in step 19.1 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Phase and baseline correction Correct the phase of the diagonal signals so that they are all negative. The NOESY correlation signals will then be positive if the compound has MW < 600 (note that this value is approximate). Any COSY artifacts will have the same phase as the diagonal signals. To simplify the phasing of the 2D NOESY spectrum, it helps to phase correct the first row: Enter rser 1 to transfer the first row to the 1D data set ~TEMP/1/1 Enter sinm to apply the sine-bell windowing function Enter ft to perform Fourier transform Manually phase correct the signals so that they are all negative (see Appendix B) to save the phase correction to the source 2D dataset to return from 1D Phase Mode to return the 2D data set Enter xfb to Fourier transform the NOESY spectrum again, this time applying the appropriate phase correction to F2 dimension The spectrum should now require additional phase correction only in F1 dimension: Expand a diagonal peak at the far left of the spectrum Right-click at the peak position and select Add to display the full spectrum

59 58 Expand a diagonal peak at the middle of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a diagonal peak at the far right of the spectrum Right-click at the peak position and select Add to display the full spectrum to phase correct columns By default, all columns are selected as indicated by the filled blue squares. To select one column: left-click in the corresponding part of the data window. To select all the columns: click on. The red vertical line indicates the default pivot point in the upper column. To change the pivot point: right-click at peak and select Set Pivot Point. Left-click-hold (zero order phase correction) and move the mouse until the phase of the reference peak of the first column is negative Left-click-hold (first order phase correction) and move the mouse until the phase of the reference peak of the second and third column is negative to save and return, or click on to return without save to return from 2D Phase Mode Enter absb to automatically correct the baseline 19.8 Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 and F1 frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined diagonal peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-1H.xwp Select Use plot limits from screen / CY Select Fill data set list with projections

60 59 NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

61 H, 1 H Correlation Through Space: ROESY, T- ROESY, and off-res-roesy ROESY (Rotating-frame Overhauser Effect SpectroscopY) is a 2D technique that gives essentially the same information as NOESY. The advantage of the ROESY experiment over the NOESY experiment is that NOE in the rotating frame under spin-lock conditions is always positive. Therefore, NOEs can be seen in ROESY spectra regardless of molecular weight. The main disadvantage of the ROESY experiment is that HOHAHA (HOmonuclear HArtmann-HAhn) correlations may also break through. However, this can be avoided using a modified version called the T-ROESY (Transverse-ROESY) pulse sequence. The off-resonance ROESY experiment can be considered a combination of the NOESY and ROESY experiments. It provides both efficient suppression of HOHAHA transfer of magnetization and reduction of offset effects. Please read Appendix C before doing these experiments! 20.1 Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Off-res-ROESY only: enter o1 and write down the value Enter sr and write down the value 20.2 Create a ROESY experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_ROESY, AN_T- ROESY or AN_off-res-ROESY Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 20.2, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 20.1 for both the F2 and F1 Frequency axis Enter the O1P value from step 20.1

62 61 Off-res-ROESY only: Click on next to FQLIST Click on next to FQ1LIST Select an existing list or create a new file Click on, a text editor opens First text row: o Second text row: the O1 value from step 20.1 Third text row: the O1 value from step Save and close the list Click on Click the ProcPars tab Enter the SR value from step 20.1 for both the F2 and F1 Frequency axis Click the Spectrum tab NOTE: The pulse P15 sets the length of the spinlock period. In the parameter sets AN_ROESY, AN_T-ROESY, and AN_off-res-ROESY the value of P15 is set to 500 ms. A good rule of thumb is that P15 for the ROESY experiment of a molecule should be the same as D8 for the NOESY experiment of that molecule. In order to set the length of the spinlock pulse, enter p15 and then enter the appropriate value Acquisition and Fourier transform Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 1 h 40 min for all three experiments Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 20.1 EXPNO the experiment number of the sample measured in step 20.1 PROCNO the processing number of the sample measured in step 20.1

63 62 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Phase and baseline correction Correct the phase of the diagonal signals so that they are all negative. Then the true ROESY cross peaks will all be positive. To simplify the phasing of the 2D ROESY spectrum, it helps to phase correct the second row: Enter rser 1 (T-ROESY, off-res-roesy) or rser 2 (ROESY) to transfer the first/second row to the 1D data set ~TEMP/1/1 Enter sinm to apply the sine-bell windowing function Enter ft to perform Fourier transform Manually phase correct the signals so that they are all negative (see Appendix B) to save the phase correction to the source 2D dataset to return from 1D Phase Mode to return the 2D data set Enter xfb to Fourier transform the ROESY spectrum again, this time applying the appropriate phase correction to F2 dimension The spectrum should now require additional phase correction only in F1 dimension: Expand a diagonal peak at the far left of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a diagonal peak at the middle of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a diagonal peak at the far right of the spectrum Right-click at the peak position and select Add to display the full spectrum to phase correct columns By default, all columns are selected as indicated by the filled blue squares. To select one column: left-click in the corresponding part of the data window. To select all the columns: click on. The red vertical line indicates the default pivot point in the upper column. To change the pivot point: right-click at peak and select Set Pivot Point. Left-click-hold (zero order phase correction) and move the mouse until the phase of the reference peak of the first column is negative Left-click-hold (first order phase correction) and move the mouse until the phase of the reference peak of the second and third column is negative to save and return, or click on to return without save

64 63 to return from 2D Phase Mode Enter absb to automatically correct the baseline 20.7 Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 and F1 frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined diagonal peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-1H.xwp Select Use plot limits from screen / CY Select Fill data set list with projections NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

65 64 21 Direct 1 H, 13 C Correlation: HMQC HMQC (Heteronuclear Multiple Quantum Coherence) is a heteronuclear 2D technique that can be used to determine which 1 H nuclei are directly ( 1 J CH ) bonded to which 13 C nuclei. Note that all correlation peaks of HMQC experiment are splitted along the F1 dimension due to evolution of the homonuclear 1 H, 1 H coupling. Therefore, the resolution of the HMQC experiment along the F1 dimension is limited. For a better resolution in the F1 dimension the HSQC experiment should be performed (see Chapter 22) Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 21.2 Acquire a standard 1D 13 C spectrum Acquire and process a standard 13 C spectrum as described in Chapter 6 (steps ) Expand the spectrum so that the 13 C signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 21.3 Create an HMQC experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_HMQC Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 21.3, you should do locking (step 3.4) and shimming (step 3.5) at this point.

66 Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 21.1 for the F2 Frequency axis ( 1 H) Enter the SW value from step 21.2 for the F1 Frequency axis ( 13 C) Enter the O1P value from step 21.1 ( 1 H) Enter the O2P value (the O1P value) from step 21.2 ( 13 C) Click the ProcPars tab Enter the SR value from step 21.1 for the F2 ( 1 H) Frequency axis and from step 21.2 for the F1 ( 13 C) Frequency axis Click the Spectrum tab NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value (this can be any number greater than 1). Check the acquisition time by clicking on Acquisition, Fourier transform and baseline correction Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 21 min Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation Enter absb to automatically correct the baseline NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the F2 ( 1 H) projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 21.1 EXPNO the experiment number of the sample measured in step 21.1 PROCNO the processing number of the sample measured in step 21.1

67 66 Repeat the above for the F1 ( 13 C) projection. This time, give the name, the experiment number, and the processing number of the sample measured in step 21.2 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 (1H) and F1 (13C) frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step 21.1 (F2 dimension) and from the 1D 13 C spectrum measured in step 21.2 (F1 dimension) Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-13C.xwp Select Use plot limits from screen / CY Select Fill data set list with projections NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

68 67 22 Direct 1 H, 13 C Correlation: HSQC HSQC (Heteronuclear Single Quantum Coherence) is a heteronuclear 2D technique that yields exactly the same information as the HMQC. Compared to the HMQC experiment no line broadening along the F1 dimension appears, as only 13 C single-quantum magnetization is present during the t 1 evolution period. Consequently, the HSQC experiment has better resolution along the F1 dimension in comparison with the HMQC experiment described in Chapter Tune and match the probe for decoupling 1 H observation and 13 C If necessary, the probehead should be tuned and matched for 1 H observation and 13 C decoupling. Follow the instructions given in Appendix D Tuning and matching 13 C Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 22.3 Acquire a standard 1D 13 C spectrum Acquire and process a standard 13 C spectrum as described in Chapter 6 (steps ) Expand the spectrum so that the 13 C signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 22.4 Create an HSQC experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_HSQC Type the dataset title in the TITLE box

69 68 NOTE: If you are starting your NMR session from step 22.4, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 22.2 for the F2 Frequency axis ( 1 H) Enter the SW value from step 22.3 for the F1 Frequency axis ( 13 C) Enter the O1P value from step 22.2 ( 1 H) Enter the O2P value (the O1P value) from step 22.3 ( 13 C) Click the ProcPars tab Enter the SR value from step 22.2 for the F2 ( 1 H) Frequency axis and from step 22.3 for the F1 ( 13 C) Frequency axis Click the Spectrum tab NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value (this can be any number greater than 1). Check the acquisition time by clicking on Acquisition and Fourier transform Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 21 min Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the F2 ( 1 H) projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 22.2 EXPNO the experiment number of the sample measured in step 22.2 PROCNO the processing number of the sample measured in step 22.2

70 69 Repeat the above for the F1 ( 13 C) projection. This time, give the name, the experiment number, and the processing number of the sample measured in step 22.3 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Phase and baseline correction Generally, the 2D spectrum is first phase corrected in the F2 dimension (rows), and then in the F1 dimension (columns). For the correction in the F2 dimension, three rows each with a cross peak should be selected. The cross peak of one row should be the far left of the spectrum, the cross peak of the second row should be close to the middle, and the one for the third row should be the far right of the spectrum. For the correction in the F1 dimension, three columns rather than rows should be selected. Expand a cross peak at the far-left-down of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a cross peak at the middle of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a cross peak at the far-right-up of the spectrum Right-click at the peak position and select Add to display the full spectrum to phase correct rows, or click on to phase correct columns By default, all rows/columns are selected as indicated by the filled blue squares. To select one row/column: left-click in the corresponding part of the data window. To select all the rows/columns: click on. The red vertical line indicates the default pivot point in the upper row/column. To change the pivot point: right-click at peak and select Set Pivot Point. Left-click-hold (zero order phase correction) and move the mouse until the phase of the reference peak of the first column is positive Left-click-hold (first order phase correction) and move the mouse until the phase of the reference peak of the second and third column is positive to save and return, or click on to return without save to return from 2D Phase Mode Enter absb to automatically correct the baseline

71 Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 (1H) and F1 (13C) frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step 22.2 (F2 dimension) and from the 1D 13 C spectrum measured in step 22.3 (F1 dimension) Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-13C.xwp Select Use plot limits from screen / CY Select Fill data set list with projections NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

72 71 23 Long-Range 1 H, 13 C Correlation: HMBC HMBC (Heteronuclear Multiple Bond Correlation) is a heteronuclear 2D technique that is suitable for determining long-range 1 H, 13 C connectivities (typically 2 J CH and 3 J CH ). There are several drawbacks associated with the HMBC experiment. For example, some correlation signals via 1 J CH can be seen in any HMBC spectrum. These erroneous cross peaks are visible as doublets along 1 H axis (F2 dimension). It is more recommendable to run the CIGAR-HMBC experiment instead of the HMBC experiment (see Chapter 24) Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 23.2 Acquire a standard 1D 13 C spectrum Acquire and process a standard 13 C spectrum as described in Chapter 6 (steps ) Expand the spectrum so that the 13 C signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 23.3 Create an HMBC experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_HMBC Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 23.3, you should do locking (step 3.4) and shimming (step 3.5) at this point.

73 Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 23.1 for the F2 Frequency axis ( 1 H) Enter the SW value from step 23.2 for the F1 Frequency axis ( 13 C) Enter the O1P value from step 23.1 ( 1 H) Enter the O2P value (the O1P value) from step 23.2 ( 13 C) Click the ProcPars tab Enter the SR value from step 23.1 for the F2 ( 1 H) Frequency axis and from step 23.2 for the F1 ( 13 C) Frequency axis Click the Spectrum tab NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value that is a multiple of 2. Check the acquisition time by clicking on Acquisition, Fourier transform and baseline correction Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 1 h 25 min Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation Enter absb to automatically correct the baseline NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the F2 ( 1 H) projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 23.1 EXPNO the experiment number of the sample measured in step 23.1 PROCNO the processing number of the sample measured in step 23.1

74 73 Repeat the above for the F1 ( 13 C) projection. This time, give the name, the experiment number, and the processing number of the sample measured in step 23.2 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 (1H) and F1 (13C) frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step 23.1 (F2 dimension) and from the 1D 13 C spectrum measured in step 23.2 (F1 dimension) Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-13C.xwp Select Use plot limits from screen / CY Select Fill data set list with projections NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

75 74 24 Long-Range 1 H, 13 C Correlation: CIGAR-HMBC There are several drawbacks associated with the HMBC experiment described in Chapter 23. First, as in the case of HMQC, 1 H multiplet structure appears along F1 as well as F2 dimension, thus limiting the 13 C resolution. Second, the HMBC experiment uses a single fixed polarization delay based on an average value of n J CH = 10 Hz. However, molecules actually have a range of n J CH values, typically from 2 to 15 Hz. Consequently, peaks arising from values of n J CH that are well removed from the average value will be significantly attenuated. Third, depending on the spin coupling constant values, a breakthrough of 1 J CH signals is observed in the HMBC experiment. These erroneous cross peaks are visible as doublets along 1 H axis (F2 dimension). In order to distinguish these signals from the desired correlations it is advisable not to use 13 C broadband decoupling. However, 13 C decoupling would be highly desirable because of the sensitivity gain obtainable by sharpening the multiplets in the F2 dimension. The CIGAR-HMBC (Constant time Inverse-detected Gradient Accordion Rescaled long-range HMBC) experiment allows the user to suppress the 1 H, 1 H coupling modulation along F1 completely to maximize resolution in the second dimension. In addition, it uses the accordion principle to sample over a range of n J CH coupling constants. Therefore, it is possible to observe more correlation signals compared to the HMBC experiment. Furthermore, due to the two-fold low-pass J-filter all 1 J CH correlation signals are effectively suppressed. Therefore, broadband decoupling can be used without the problem of ambiguity between 1 J CH and n J CH correlations Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 24.2 Acquire a standard 1D 13 C spectrum Acquire and process a standard 13 C spectrum as described in Chapter 6 (steps ) Expand the spectrum so that the 13 C signals cover almost the entire spectral width Enter sw and write down the value Enter rg and write down the value Enter o1p and write down the value

76 75 Enter sr and write down the value 24.3 Create a CIGAR-HMBC experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_CIGAR-HMBC Type the dataset title in the TITLE box NOTE: If you are starting your NMR session from step 24.3, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 24.1 for the F2 Frequency axis ( 1 H) Enter the SW value from step 24.2 for the F1 Frequency axis ( 13 C) Enter the RG value from step 24.2 Enter the O1P value from step 24.1 ( 1 H) Enter the O2P value (the O1P value) from step 24.2 ( 13 C) Click the ProcPars tab Enter the SR value from step 24.1 for the F2 ( 1 H) Frequency axis and from step 24.2 for the F1 ( 13 C) Frequency axis Click the Spectrum tab NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value that is a multiple of 2. Check the acquisition time by clicking on Acquisition, Fourier transform and baseline correction Press [SPIN ON-OFF] to stop spinning Enter zg to start the acquisition; the expected experiment time is ca. 1 h 30 min Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation Enter absb to automatically correct the baseline NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of

77 76 *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the F2 ( 1 H) projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 24.1 EXPNO the experiment number of the sample measured in step 24.1 PROCNO the processing number of the sample measured in step 24.1 Repeat the above for the F1 ( 13 C) projection. This time, give the name, the experiment number, and the processing number of the sample measured in step 24.2 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 (1H) and F1 (13C) frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step 24.1 (F2 dimension) and from the 1D 13 C spectrum measured in step 24.2 (F1 dimension) Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-13C.xwp Select Use plot limits from screen / CY Select Fill data set list with projections

78 77 NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

79 78 25 Long-Range HSQMBC 1 H, 13 C Correlation: G-BIRD- The G-BIRD-HSQMBC (Heteronuclear Single Quantum Multiple Bond Correlation) experiment provides pure absorption, antiphase lineshape for precise, direct measurement of n J CH coupling constants. The very clean data provided by this experiment generally allow one to measure accurately coupling constants directly from F2 ( 1 H) slice through the HSQMBC correlation of interest. See R. T. Williamson et al., Magn. Reson. Chem., 38 (2000) 265 and B. L. Marquez et al., Magn. Reson. Chem., 39 (2001) 499 for details and examples Acquire a standard 1D 1 H spectrum Acquire and process a standard 1 H spectrum as described in Chapter 5 (steps ) Expand the spectrum so that the 1 H signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 25.2 Acquire a standard 1D 13 C spectrum Acquire and process a standard 13 C spectrum as described in Chapter 6 (steps ) Expand the spectrum so that the 13 C signals cover almost the entire spectral width Enter sw and write down the value Enter o1p and write down the value Enter sr and write down the value 25.3 Create a G-BIRD-HSQMBC experiment (edc) Enter desired value for NAME and EXPNO Click the down-arrow of the Solvent box and select a solvent Click the down-arrow of the Experiment box and select AN_G-BIRD- HSQMBC Type the dataset title in the TITLE box

80 79 NOTE: If you are starting your NMR session from step 25.3, you should do locking (step 3.4) and shimming (step 3.5) at this point Set the acquisition and processing parameters Click the AcquPars tab Enter the SW value from step 25.1 for the F2 Frequency axis ( 1 H) Enter the SW value from step 25.2 for the F1 Frequency axis ( 13 C) Enter the O1P value from step 25.1 ( 1 H) Enter the O2P value (the O1P value) from step 25.2 ( 13 C) Click the ProcPars tab Enter the SR value from step 25.1 for the F2 ( 1 H) Frequency axis and from step 25.2 for the F1 ( 13 C) Frequency axis Click the Spectrum tab NOTE: It is advisable to use linear prediction to improve the resolution in F1 dimension (see Appendix E). NOTE: It may be necessary, depending on your sample concentration, to run the experiment with higher number of scans. In order to do this, enter ns and then enter a new higher value that is a multiple of 8. Check the acquisition time by clicking on Acquisition and Fourier transform Press [SPIN ON-OFF] to stop spinning Enter rga to automatically set the receiver gain Wait for the rga: finished message Enter zg to start the acquisition; the expected experiment time is ca. 3 h 10 min Wait for the zg: finished message Enter xfb to perform the 2D Fourier transformation NOTE: Using the command xfb it is possible to peek at the accumulated data at its current state before acquisition terminates. NOTE: Left-click-hold the 2D spectrum. and move the mouse to increase/decrease the intensity of *If you are performing processing on your own PC, you can skip steps * D projections Right-click inside the F2 ( 1 H) projection area of the data window Select External Projection Select Display data in same window Change the following parameters: NAME the name of the sample measured in step 25.1

81 80 EXPNO the experiment number of the sample measured in step 25.1 PROCNO the processing number of the sample measured in step 25.1 Repeat the above for the F1 ( 13 C) projection. This time, give the name, the experiment number, and the processing number of the sample measured in step 25.2 NOTE: To change the intensity of the 1D projections: left-click the projection to select it (blue square is filled) and left-click-hold and move the mouse to increase/decrease the intensity Phase and baseline correction Generally, the 2D spectrum is first phase corrected in the F2 dimension (rows), and then in the F1 dimension (columns). For the correction in the F2 dimension, three rows each with a cross peak should be selected. The cross peak of one row should be the far left of the spectrum, the cross peak of the second row should be close to the middle, and the one for the third row should be the far right of the spectrum. For the correction in the F1 dimension, three columns rather than rows should be selected. Expand a cross peak at the far-left-down of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a cross peak at the middle of the spectrum Right-click at the peak position and select Add to display the full spectrum Expand a cross peak at the far-right-up of the spectrum Right-click at the peak position and select Add to display the full spectrum to phase correct rows, or click on to phase correct columns By default, all rows/columns are selected as indicated by the filled blue squares. To select one row/column: left-click in the corresponding part of the data window. To select all the rows/columns: click on. The red vertical line indicates the default pivot point in the upper row/column. To change the pivot point: right-click at peak and select Set Pivot Point. Left-click-hold (zero order phase correction) and move the mouse until the phase of the reference peak of the first column is positive Left-click-hold (first order phase correction) and move the mouse until the phase of the reference peak of the second and third column is positive to save and return, or click on to return without save to return from 2D Phase Mode Enter absb to automatically correct the baseline

82 Calibration Calibration is performed automatically according to the SR parameter. If the automatic calibration does not give a satisfactory result, calibration has to be done manually: Expand the reference peak Move the red crosshair at the reference peak and left-click Enter the F2 (1H) and F1 (13C) frequency you want to assign to the reference peak NOTE: Select an intensive and well-defined peak for the reference. Check its chemical shift from the 1D 1 H spectrum measured in step 25.1 (F2 dimension) and from the 1D 13 C spectrum measured in step 25.2 (F1 dimension) Measurement of coupling constants Zoom in a correlation peak of interest Select Processing/Rows and columns Select Interactive row/column display, the button turns green Move the mouse in the data window to find the maximum intensity for the peak of interest and right-click Select Extract Row/Column Enter desired value for PROCNO and click on. The extracted row is stored as a 1D dataset under the specified PROCNO and displayed in a new data window to shift the zero line of the 1D spectrum to the center of the screen Zoom in a peak of interest, the button turns green Left-click-hold at one peak position and drag the mouse to another peak position; the distance in Hz will be displayed Right-click in the data window to exit distance measurement mode Printing Define the print region using the mouse Select Print with layout plot directly Select LAYOUT= +/AN_2D_1H-13C.xwp Select Use plot limits from screen / CY Select Fill data set list with projections

83 82 NOTE: Left-click-hold and move the mouse to increase/decrease the intensity of the 2D spectrum. Left-click-hold and move the mouse to change the level distance. Click on to store contour levels. Clicking on resets the intensity to the last saved intensity. NOTE: To change the intensity of the 1D projections in the printout, the TopSpin Plot Editor must be used. See Appendix B for instructions.

84 APPENDIXES 83