Introduction to 1D & 2D NMR using VnmrJ

Transcription

1 Introduction to 1D & 2D NMR using VnmrJ Scott R. Burt September 12, 2011 This document is an introductory tutorial for collecting the most important NMR spectra in organic chemistry: (1D) 1 H, 13 C, NOESY, (2D) COSY, HSQC, and HMBC. The document begins with a brief outline of the steps required to prepare a sample, setup the instrument, collect the data, and prepare the spectra for printing. The remainder of this document contains a detailed review of these steps, addressing the most common problems and questions that face new users of the NMR facility. More information can be found in the Agilent VnmrJ Quickstart Guide and the Agilent VnmrJ Experiment Guide. Please report any errors to Dr. Burt. Contents 1 Quick Reference Checklist for Acquiring NMR Data Checklist for Processing and Printing NMR Data Recommendations and Things to Remember Command Summary and VnmrJ Locations Software Overview Instrument Access RedHat Linux Operating System VnmrJ Interface Preparing Your Sample Guidelines for Sample Preparation Initial Instrument Setup Acquiring Your Data Creating a Study Customizing the Study Submitting the Study for Acquisition Processing & Printing Your Data Processing Printing

2 QUICK REFERENCE Intro to 1D & 2D NMR 1 Quick Reference 1.1 Checklist for Acquiring NMR Data 1. Prepare your sample: Your sample should have a concentration of mm, see table 1. Use µl of solvent. The ideal solvent height is 5 cm (2 in). Use a pure, dry, deuterated solvent. Suspended particles, bubbles, or emulsions will degrade the spectral quality. 2. Log in to VnmrJ: Use your department network account. If you are using the instrument for a class, use the account info given to you. 3. Place your sample in the magnet: (a) Eject the sample in the magnet. (b) Remove the sample from the turbine and place it in the sample holder. (c) Place your sample in the turbine using the depth-gauge. (d) Insert your sample in the magnet. 4. Load the default shims: (fig. 3A) 5. (500 MHz only) Match and tune the probe. 6. Create a new study: (a) Click on the new study button. (b) Enter the sample name. This name is used when auto-saving your data. (c) Select the deuterated solvent you used to prepare your sample. (d) (Optional) Enter descriptive text in the comment box. This information will be included on your final print out. 7. Customize the study: (a) Select the experiments you want to run from the experiment selector. (b) Customize the experiments by double-clicking on the experiment nodes. The Agilent VnmrJ Experiment Guide describes the available options. (c) (Optional) Submit the initial study using only a PROTON experiment. If the purity and concentration are acceptable; use continue study, turn off lock and shim, and customize the rest of the study. 8. Submit the study for acquisition. 9. When you are finished collecting your data, repeat step 3 to remove your sample and place the flame-sealed standard sample back in the magnet. (sec. 3.2) 10. Log out of VnmrJ when you are finished. (fig. 3A) 2 BYU NMR Facility

3 Intro to 1D & 2D NMR QUICK REFERENCE 1.2 Checklist for Processing and Printing NMR Data 1. Use the data processing computers to process and print your data. 2. Open a data set: (a) Load a study into the study queue using the File, Open... menu option. Navigate to the desired study directory, select it and click Open. (b) Double-click on an experiment node in the study queue or drag the experiment node to the graphics canvas to open and auto-process that data set. (c) Occasionally, experiment nodes fail to load when double-clicked or dragged to the graphics canvas. If this happens, open one of the experiments inside of the study folder and then type rebuildxml on the command line to fix this problem. 3. Process the data: (a) Use the process tab, default page. (b) Adjust the spectrum display using the left, right and middle mouse buttons (including the scroll-wheel) in addition to the zoom in, zoom out, and pan/stretch tool buttons. (c) (Optional) Adjust the spectral referencing to TMS, the solvent, or manually reference the spectrum to a peak with a known chemical shift. (d) 1D Specific Processing (Optional) Adjust the phase if auto-process failed to phase the spectrum. ( 1 H) Set the threshold and use auto integrate to set the integration regions. ( 1 H) Use the scissor tool (integral resets) to adjust the integral reset points and then normalize the integral values. ( 1 H) Set the two cursors on different peaks in a multiplet and type delta? in the command line to measure the splitting in Hz. ( 13 C) Set the threshold for printing peak frequencies or peak lists. (e) 2D Specific Processing Use the increase/decrease vertical scale buttons or the middle scroll-wheel to adjust the cut-off threshold. (f) Save your processing with the save current process/display parameters button. 4. Print the data: (a) Use the process tab, plot page. (b) Adjust the various plot options (e.g., parameters) using the drop-down menus. Choose none to prevent a particular option from printing. (c) (1D) Preview integrals and/or peak frequencies using the show buttons. (d) (2D) Adjust the side-spectra options and plot size if desired. (e) Convert the spectrum to a PDF file using autoplot preview. (f) Print the spectrum using automatic plot page or use the send to plotter option in the plot view window. BYU NMR Facility 3

4 QUICK REFERENCE Intro to 1D & 2D NMR 1.3 Recommendations and Things to Remember The following equations and tables are discussed in more detail in sec. 3.1 and sec. 4.2; they are included here for quick reference. Table 1 gives the minimum concentration required to obtain quality spectra using the default parameters. The time required using the default settings is also given. Table 1: Recommended Minimum Concentrations Experiment Time 300 MHz 500 MHz 1D 1 H 30 s 10 mm 5 mm gcosy 5 min 16 mm 9 mm HSQCAD 8 min 22 mm 12 mm ghmbcad 32 min 22 mm 12 mm 1D 1 3C 34 min 60 mm 20 mm DEPT (full) 18 min 60 mm 20 mm DEPT (135 ) 5 min 60 mm 20 mm Use eqn. 1 to estimate the number of milligrams needed for a desired concentration or to estimate the concentration from the number of milligrams used: W # mg in 1 ml 10 mm (1) 100 W is the molar mass in g/mol. As shown in eqn. 2, the signal-to-noise ratio (SNR) is proportional to both the concentration, C, and the number of scans, n (or acquisition time, t). This allows one to use table 1 and eqn. 3 to estimate the time needed for a sample with a particular concentration. SNR C n SNR C t (2) C 1 n1 = C 2 n2 C 1 t1 = C 2 t2 (3) 2D NMR requires an appropriate number of t1 increments to avoid ringing artifacts and to obtain sufficient resolution along the F1 axis. Tables 2 and 3 provide some guidance for selecting the number of t1 increments. Table 2: Recommended number of t1 increments for 1 H 1 H experiments (COSY, TOCSY, etc.) Resolution 300 MHz 500 MHz Notes Low Good for simple spectra, may get some ringing Default Default values High Necessary for crowded spectra Table 3: Recommended number of t1 increments for 13 C 1 H experiments (HSQC, HMBC, etc.) Resolution 300 MHz 500 MHz Notes Low Good for simple spectra, may get some ringing Default Default values High Necessary for crowded spectra 4 BYU NMR Facility

5 Intro to 1D & 2D NMR QUICK REFERENCE 1.4 Command Summary and VnmrJ Locations Some users find the command-line more convenient than the point-and-click interface. The most useful commands are summarized below. Table 4: VnmrJ commands and locations Command VnmrJ Button Action Performed Setup (Start Tab) e Eject Eject the sample. i Insert Insert the sample. sload Load Default Shims Load the most recently calibrated shim file. Acquisition (Acquire Tab) nt=# Default Number of Scans (per t1 Increment) Number of scans to acquire during the experiment (or during each t1 increment for 2D experiments.) ni=# Default Number of t1 Increments Number of points to acquire in the indirect dimension of a 2D experiment. bs=# Flags Scans per block How often to send acquired data to computer. time Show Time Calculate and display how long experiment will last. Processing (Process Tab Default) process Autoprocess Automatically process and display data. wft Transform Calculate and display weighted Fourier transform. aph Autophase Automatically phase the spectrum. vsadj Autoscale Automatically adjust spectrum height. cz Clear Integrals Clear all integral resets. ds Display Spectrum Display spectrum without recalculating. f Full Spectrum Display entire spectral width. full Full Screen Force display window to full size. nl Find Nearest Line Move cursor to the top of the nearest peak. rl(#p) Reference Cursor to # Change how the scale is referenced. res Reports line shape parameters of the selected peak. delta? Reports difference, in Hz, between the two cursors. (Along F2 for 2D spectra) delta1? (2D spectra only) Reports difference along F1, in Hz, between the two cursors. Workspace explib Workspace Menu Open workspace interface to manage experiments. cexp(#) Workspace Menu Create a new experiment, exp#. delexp(#) Workspace Menu Delete experiment #. jexp# Workspace Menu Join experiment #. unlock(#) Unlock experiment #. Use if you get an experiment locked error or foreground processing active error. mf(#,#) Edit Move FID Copy all parameters and data from one experiment to another. BYU NMR Facility 5

6 SOFTWARE OVERVIEW Intro to 1D & 2D NMR 2 Software Overview 2.1 Instrument Access Only users who have been trained may use the instrument. If you have a research account, log in using your department network account. Your advisor must add your account to one of his/her network groups. Undergraduate network accounts expire every semester. Graduate network accounts expire every year. Renew your password in the CSR office or in the main computer lab. If you are using the instrument for a class, you will share a class account. A username and password will be given to you during the initial training. 2.2 RedHat Linux Operating System The desktop environment is shown in figure 1. You may use USB drives to backup your data. To eject the USB drive, right-click the USB icon and select unmount volume. The computers have a number of useful programs installed including a web-browser, the Open Office suite and the Adobe PDF reader. The web-browser points to the scheduling page by default, fig. 1A. Your NMR data are saved to the network drive. Access your data by opening the home folder and then the vnmrdata folder; your NMR data folder will be found here. fig. 1B. Figure 1: Linux desktop. A) scheduling page shortcut (web browser), B) home folder containing a link to the network drive (vnmrdata) where all the NMR data is stored. 6 BYU NMR Facility

7 Intro to 1D & 2D NMR PREPARING YOUR SAMPLE 2.3 VnmrJ Interface The following toolbars and panels may be hidden when you first log in to VnmrJ; use the view menu to reveal hidden panels and toolbars, fig. 2A. Tool bars these icons help you open studies, load the default shims, auto-integrate peaks and log out of the current operator, fig. 3A. Vertical panels the Protocol vertical panel allows you to create and view NMR studies, fig. 3B. The Frame and Viewport vertical panels provide more advanced data analysis and processing features, see the Advanced 1D & 2D NMR Tutorial for details. The command line enter text commands here, fig. 3C and table 4. Tabs and pages these panels provide tools for setting up the instrument and configuring experiments as well as tools for processing and printing, fig. 3D. Graphics canvas this window displays your spectrum, fig. 3E. Dragging experiment nodes here will load and auto-process the data. Graphics controls these buttons are used to process the data, fig. 3F. If you hover the mouse cursor over these icons, a descriptive label will appear. 3 Preparing Your Sample 3.1 Guidelines for Sample Preparation The quality of your NMR data reflects the quality of your sample. The following guidelines will make it easier to lock, shim and interpret your NMR data. Use a clean, high quality NMR tube. For high resolution spectra, use an NMR tube with the same frequency rating as the magnet, e.g. 300 MHz. Using NMR tubes rated for a higher frequency will not improve your spectra and are simply a waste of money. For quick and dirty spectra, it is fine to use economy NMR tubes (typically rated for 60 MHz.) Your NMR tube should be perfectly straight. Warped NMR tubes will damage the instrument and may break inside the magnet. A straightness-tester is placed by each depth-gauge if you want to test your NMR tube. When re-using NMR tubes, do not place the NMR tubes in a flask or beaker and dry them at high temperatures in a drying oven. Instead, lay the tubes flat on a paper towel in a flat metal tray that is raised on one end. Do not use temperatures exceeding 150 C. Rinse your tubes with D 2 O or another dueterated solvent. Ensure that the outside of your NMR tube is clean. Grease and other chemicals will build up inside the magnet, resulting in background signals and potentially damaging expensive electronics. BYU NMR Facility 7

8 PREPARING YOUR SAMPLE Intro to 1D & 2D NMR Figure 2: View menu. A) open panels, toolbars, command line, etc.; B) hide or close a panel. Figure 3: VnmrJ interface. A) system and user toolbars, B) vertical panels, C) command line, D) tabs and pages, E) graphics canvas, F) graphics controls. 8 BYU NMR Facility

9 Intro to 1D & 2D NMR PREPARING YOUR SAMPLE Use a pure, dry, deuterated solvent. Suspended particles, bubbles, or emulsions are difficult to lock and shim and will reduce both the resolution and the SNR of your spectrum. Mixed solvents are usually fine, but they may confuse the auto-shimming and auto-referencing routines, requiring manual shimming and referencing. (Optional) Use a solvent with TMS pre-added or add a small amount of TMS to your sample. The TMS peak is a great way to check your lineshape for shimming artifacts. TMS provides an internal reference which can be more accurate than using the solvent for referencing. TMS is not soluble in D 2 O; instead, use a single crystal of DSS. TMS and TMS-based referencing can be problematic when using silyl protecting groups. Use a sample height of approximately of 5 cm ( ml). Short samples are difficult to shim, resulting in poor resolution and SNR. Concentrating your sample by reducing the sample volume will not improve the quality of your spectrum. A well shimmed short sample will have a lower SNR than the same sample diluted to a 5 cm height. Choose a good concentration: A concentration of mm will generally work well. Table 5 gives the minimum concentration required to obtain quality spectra using the default experiment parameters. Lower concentrations require an increase in signal averaging time according to eqn 4: C 1 n1 = C 2 n2 (4) where C is the concentration and n is the number of scans. Estimate the number of mg that you need using equation 5: W # mg in 1mL 10mM (5) 100 Dividing the molar mass, W (in g/mol), by 100 gives you the number of mg that are needed in 1 ml of solution to give 10 mm. Scale this number of mg to get the desired concentration. Some molecules may aggregate at higher concentrations while other molecules may increase the viscosity of the solution at high concentrations; both situations lead to severe degradation of the spectral quality. Do not saturate the solution; changes in temperature may lead to precipitation of your solute and solid particles in the NMR tube will degrade the quality of your spectra. BYU NMR Facility 9

10 ACQUIRING YOUR DATA Intro to 1D & 2D NMR Table 5: Recommended Minimum Concentrations Experiment Time 300 MHz 500 MHz 1D 1 H 30 s 10 mm 5 mm gcosy 5 min 16 mm 9 mm HSQCAD 8 min 22 mm 12 mm ghmbcad 32 min 22 mm 12 mm 1D 1 3C 34 min 60 mm 20 mm DEPT (full) 18 min 60 mm 20 mm DEPT (135 ) 5 min 60 mm 20 mm 3.2 Initial Instrument Setup The NMR instrument is very expensive and can be easily damaged by careless behavior. Please follow these guidelines when placing your sample in the magnet: 1. Choose the start tab, standard page and eject the sample that is currently in the magnet, fig. 5B. 2. Handle the turbine as shown in fig. 4A. Do not touch the shaft of the turbine; finger grease on the turbine builds up inside the spinning mechanism. 3. Carefully remove the sample from the turbine, placing the sample in the sample holder by the computer. 4. Kim wipes are provided so you can clean the bottom of your NMR tube before placing your sample in the turbine. Wipe the shaft of the turbine to ensure it is clean. 5. Carefully place your sample in the turbine using the depth-gauge to position your NMR tube, fig. 4B. 6. If your sample height is shorter than 5 cm, center the liquid around the active RF region, fig. 4D. 7. Place the turbine back in the magnet lift and insert your sample, fig. 5B. 8. Load the default shims using load std shims button in the user toolbar, fig. 3A. 4 Acquiring Your Data 4.1 Creating a Study 1. (500 MHz only) Optimize the match and tune capacitors for both the 13 C and 1 H channels; see the Advanced 1D & 2D NMR Tutorial for instructions. 2. Create a new study or open an old data set and continue this study, fig. 6B. When continuing a study, skip to sec. 4.2 and customize the study. 10 BYU NMR Facility

11 Intro to 1D & 2D NMR ACQUIRING YOUR DATA Figure 4: Sample handling. A) turbine with sample tube, B) depth-gauge with turbine and sample, C) active region in RF probe, D) properly centered short sample, E) ideal sample height. BYU NMR Facility 11

12 ACQUIRING YOUR DATA Intro to 1D & 2D NMR Figure 5: Start tab, standard page. This panel changes slightly when modifying a study. A) logout current operator, B) insert/eject sample, C) study name (required), D) select solvent, E) information about the sample and experiment, F) automatic lock/shim status. Figure 6: Study queue and experiment panels. A) experiment nodes in a completed study, B) create a new study or continue a previous study, C) add experiment nodes to the study, D) experiment nodes in the study, E) cancel any edits to this study, F) submit the study for acquisition, (optional) choose foreground or background submission, G) delete experiment nodes by dragging them to the trash. 12 BYU NMR Facility

13 Intro to 1D & 2D NMR ACQUIRING YOUR DATA 3. (Optional) Begin with just a simple PROTON (enable the calibrate pw90 option, fig. 7E). After reviewing the quality of this initial PROTON spectrum, continue the study with the remaining experiments of interest. 4. (Optional) When continuing a study, auto locking and shimming are automaticall turned off, fig. 5F. Re-enable these options only if you have removed your sample from the manget and then returned later and placed the sample back in the magnet without manually locking and shimmind. 5. Give the study a name, fig. 5C. This is used to create the study directory in which your data is auto-saved. 6. Select the solvent, fig. 5D. If the wrong solvent is selected, automatic referencing will fail and artifacts such as folding or wrapping may corrupt your spectrum. 7. (Optional) Include additional information about your sample or the experiments in the comment box, fig. 5E. This text is included on your final print out. 4.2 Customizing the Study 1. Add experiment nodes to your study using the buttons in the experiment panel, fig. 6C. The common tab has the most useful experiments. Refer to the Agilent VnmrJ Experiment Guide for more information. 2. Modify the experiment nodes in the study queue by dragging them. Dragging a node within the list, fig. 6D, will change the order and dragging a node to the trash icon, fig. 6G, will delete that node. 3. Customize the experiment parameters by double-clicking on the experiment nodes, fig. 6D. This opens the acquire tab, fig. 7, but you may have to select the default page to see the most useful options. Fig. 7 provides examples of the PROTON and HSQC panels; most 1D and 2D panels are similar to these. 4. The generic parameters that you can customize for every experiment are described below: Spectral width, fig. 7B: The spectral width must be large enough to contain all of your peaks. Any peaks that fall outside the spectral window will wrap around and appear at spurious chemical shift values. Number of scans, fig. 7C: The SNR depends on both the concentration and the number of scans as shown in eqn. 6: SNR C n (6) For example, you must increase the number of scans by a factor of 100 to see an increase in SNR by a factor of 10. This exponential increase in signal averaging time limits NMR to relatively concentrated samples, particularly for 13 C NMR. Table 5 recommends minimum concentrations when using the default number of scans. BYU NMR Facility 13

14 ACQUIRING YOUR DATA Intro to 1D & 2D NMR Show Time button, fig. 7A: This updates the estimated time on the experiment node after modifying parameters. Auto-actions, fig. 7G: Enable these to automatically re-shim the magnet before this specific experiment is run and/or automatically plot the spectrum after this experiment finishes. 5. Customize the PROTON experiment in your study queue: (a) Enable calibrate pw90, fig. 7E, to ensure that your pulses are calibrated. This reduces some artifacts and maximizes the SNR. This option is particularly important if you will run any 2D NMR experiments on your sample. (b) Set minimize SW to auto, fig. 7E, if you will run a homonuclear 2D experiment (e.g. COSY). This option will maximize the resolution in the 2D spectrum. (c) Relaxation delay, pulse angle and receiver gain, fig. 7D: These parameters affect the SNR of quaternary carbons and the quality of integral values for PROTON spectra. However, it is best to simply use the default values unless you know how to optimize these. 6. Customize the NOESY1D experiment in your study queue: (a) Run a PROTON experiment first and then continue the study before adding and configuring NOESY1D to the study queue. See sec. 4.1, step 2, for a discussion of turning off the auto lock and shim when continuing a study. (b) Place the left and right cursors around the peak of interest, fig. 8A and choose select, fig. 8C. (c) You can create a list of peaks to invert by selecting several peaks in turn. show list will display the current list of selected peaks on the graphics canvas and clear will delete all selected peaks, fig. 8C. (d) The mix=0 option, fig. 8B, runs a dummy scan that allows you to check the quality of your selective inversion profile. (e) The optimal mixing time and number of scans is highly dependent on how rigid the molecule is, the viscosity of the solvent, how quickly the molecule is tumbling in solution, and the strength of the main magnetic field. You may need to experiment with a variety of mixing times and sample conditions to obtain the best results. 7. Customize a 2D NMR experiment in your study queue: (a) Set the scans per t1 increment, fig. 7C, to a value appropriate for your concentration. This parameter is identical to the number of scans discussed in sec. 4; it improves the SNR and removes some artifacts from the spectrum. (b) Set the t1 increments, fig. 7I, to a value appropriate for your molecule. This parameter improves the resolution in the F1 dimension, removes ringing artifacts and improves the SNR (to some extent). Tables 6 and 7 give recommendations based on the complexity of your molecule. 14 BYU NMR Facility

15 Intro to 1D & 2D NMR ACQUIRING YOUR DATA Figure 7: Acquire tab, default page. A) calculate experiment time, B) spectral width, C) number of scans to average, D) parameters that affect the steady state signal, E) auto-calibration options for optimizing future 2D NMR experiments, F) pre-amplifier settings, G) automatically re-shim before this experiment starts and/or auto-plot after this experiment is done, H) choose the X nucles (e.g., 13C, 31P, 15N), the default is 13C, I) number of t1 increments, J) optional settings. Figure 8: NOESY1D setup and processing. A) select the peak of interest with a cursor window, B) choose the NOE mixing time and enable/disable a test of the inversion profile, C) tools for selecting the peak(s) of interest, D) selective inversion profile (mixing time of 0 ms), E) NOESY spectrum. BYU NMR Facility 15

16 PROCESSING & PRINTING YOUR DATA Intro to 1D & 2D NMR Table 6: Recommended number of t1 increments for 1 H 1 H experiments (COSY, TOCSY, etc.) Resolution 300 MHz 500 MHz Notes Low Good for simple spectra, may get some ringing Default Default values High Necessary for crowded spectra Table 7: Recommended number of t1 increments for 13 C 1 H experiments (HSQC, HMBC, etc.) Resolution 300 MHz 500 MHz Notes Low Good for simple spectra, may get some ringing Default Default values High Necessary for crowded spectra 4.3 Submitting the Study for Acquisition Clicking on the submit button, fig. 6F, will submit your study for acquisition. (Optional) Choose to submit to foreground or background, fig. 6F. Submitting to foreground will display the progress of the acquisition on the computer screen; however, the current experiment workspace will be locked. Submitting to background allows you to process data in the current experiment workspace while the study is acquired, but the current progress is not displayed in the graphics canvas. (Optional) Use the done button, fig. 6E, to cancel the study you have created or modified rather than submit it for acquisition. If you need to cancel the current acquisition after you have submitted it, you must use the tools in the automation menu. If you submitted your study to foreground, use foreground acquisition, pause NOW. If you submitted to background, use background acquisition, stop-save. 5 Processing & Printing Your Data In order to interpret your spectra correctly, you must be able to process and print your data correctly. The following sections describe the tools that will help you with this task. 5.1 Processing The processing tools (fig. 10) are located on the toolbar and in the process tab. The most useful tools are located in the default page Opening & Saving Data Opening an.fid file auto-process 1 and display the data. 1 Auto-processing performs Fourier-transformation, auto phase correction, auto scaling of the peak heights, zero-filling and apodization. 16 BYU NMR Facility

17 Intro to 1D & 2D NMR PROCESSING & PRINTING YOUR DATA Figure 9: File browser window. A) current location, B) a study folder, selected and ready to open, C) navigate to outer folder, D) shortcut to the network folder. Figure 10: Graphic control tools and processing tab, default page. A) actions of the various graphic control buttons, B) basic processing, C) display controls, D) spectrum reference controls, E) integration controls F) save processing and display parameters, G) referencing tools for 2D spectra. BYU NMR Facility 17

18 PROCESSING & PRINTING YOUR DATA Intro to 1D & 2D NMR If your study is already active, (i.e., it is displayed in the study queue panel, fig. 6A) you may double-click on the desired experiment node or drag the experiment node to the graphics canvas, fig. 3E, to open and auto-process the data. To open a study, use the File, Open... menu option or the Open... menu button to open the file browser, fig. 9. After selecting your study directory, use the open button to load your study into the study queue panel. The default location for the file browser is your data directory. If this is not where the file browser is pointing, use the home shortcut button, fig. 9D, to return to the network drive and then select your folder. Experiment nodes occasionally fail to load when double-clicking or dragging them to the graphics canvas. If this happens, you can use the file browser to descend into the study directory and open the.fid of interest (e.g. Proton). After opening the experiment, type rebuildxml on the command line, fig. 3C, to fix the problem. After processing your data, save your changes to the study directory using the save current process/display parameters button, fig. 10F. If you fail to save your processing parameters, you will need to re-set your integrals and referencing the next time you open this experiment node Basic Controls for Both 1D & 2D Use all three mouse buttons and the graphics control buttons 2 on the right side of the graphics canvas to zoom in/out and adjust the vertical scale of the spectrum: Left-click moves the cursor or the left edge of a cursor-window to the mouse position (lower-left edge for 2D spectra). Right-click places the second cursor at the mouse position, creating a cursorwindow or adjusting the size of an existing cursor-window (upper-right edge for 2D spectra). After setting the cursor window, the (+) magnifying glass tool will zoom to the region in the cursor window. The (-) magnifying glass tool will jump to the previous zoom setting. The full spectral width tool will zoom out to the full spectral width. Middle-click 3 adjusts the height of the spectrum. Scroll-wheel also adjusts the height of the spectrum. The pan/stretch button will let you quickly adjust the position of the current view (left-click and drag) as well as the horizontal scale (right-click and drag). For 1D spectra, you can measure J values by setting the two cursors on different peaks of a multiplet and typing delta? on the command line. The separation, in Hz, between the peaks is reported. For 2D spectra, delta? returns the frequency separation along the F2 axis while delta1? returns the frequency separation along the F1 axis. 2 Hover the mouse cursor over a tool button and a description will appear. 3 Press the scroll wheel down like a button to middle-click. 18 BYU NMR Facility

19 Intro to 1D & 2D NMR PROCESSING & PRINTING YOUR DATA Use the referencing tools to correct the frequency axis, fig. 10D. By TMS assumes the right-most peak, near 0 ppm, is TMS. 4 By solvent calculates the reference using the lock frequency. This is the new IUPAC standard, but it may differ from TMS referencing slightly. Reference cursor allows you to force a known peak to have a specific chemical shift value. For example, the residual solvent peak may be used to reference the spectrum. 2D spectra allow you to reference the F1 axis and the F2 axis either by solvent or manually (by cursor), fig. 10G D Specific Processing In order to print peak lists, peak frequencies or to use the auto integrate tool, you need to set the threshold to discriminate between undesired peaks and those of interest. The threshold button reveals a yellow threshold line that you position with the left mouse button. Any peaks below this threshold line will be ignored. Auto-phasing does not always work perfectly. Use the following procedure to manually phase your spectrum: Turn on phase mode by pressing the phase button. Left-click on the tallest peak in your spectrum. Click and hold the left mouse button. Drag the cursor up and down to change the phase of the peak; adjust the peak so it has a flat baseline. Left-click another peak that is as far away from the first peak as possible. Adjust the phase of this peak as described above. You may need to repeat this several times. You may need to increase the scale and/or zoom in to the peaks of interest before adjusting the phase. Several tools are available to help you integrate your spectrum, fig. 10E: Begin with the clear integrals button or set the threshold and use the auto integrate tool, fig. 3A. The integral mode button cycles between part integral, full integral and no integral. It is often convenient to use a large vertical scale when setting your integral resets. Turn integration mode off in order to change the vertical scale of your spectrum. Define peaks or regions of multiple peaks by setting integral resets (the tool button with a picture of scissors). The integral resets button only appears when part integral mode is active. In integral reset mode, left-click on the spectrum to place an integral reset or right-click to remove an integral reset. 4 This may fail when using certain protecting groups that have negative chemical shifts such as TBS. BYU NMR Facility 19

20 PROCESSING & PRINTING YOUR DATA Intro to 1D & 2D NMR Normalize the integral values by placing a cursor on an integrated peak that you know has 1, 2, or 3 protons. Enter the number of protons in the text box under integration and press the set norm to button. 5 Preview the integral values with the integral values button. You may need to adjust the vertical position of the spectrum to see the integral values (see below). (Optional) setting the norm to 100 and using normalized integrals displays integrals as percentages of the total integrated area. This is useful for analyzing mixtures of different compounds. Middle-click and the scroll-wheel adjust the vertical scale of the spectrum unless integration mode is turned on, then the vertical scale of the integral regions are adjusted instead. Middle-clicking at the left-most edge of the spectrum will change the vertical position of the spectrum; this may be necessary for displaying or printing integral values. If part integral mode is turned on, middle-clicking at the left-most edge of the spectrum will change the vertical position of the integral resets. Measure the lineshape of a peak by typing res on the command line. This command automatically analyzes the tallest peak in the current view, so you may have to zoom in on the peak of interest NOESY1D Specific Processing Processing NOESY1D spectra is very similar to processing 1D proton spectra, but there are some differences. The tips below will help you. If mix=0 was enabled, the spectrum that you first see is simply your selective inversion profile. Use this to check for accidental inversion of undesired peaks as well as STP artifacts. To get to the actual NOESY spectrum, use the next spectrum button, fig. 10A. If you ran a series of mixing times, the next spectrum and previous spectrum buttons will move between the different spectra in the array. The auto-phasing for NOESY1D is rarely perfect (largely due to SPT artifacts). Increase the vertical scale so the NOE peaks are clearly visible. Start by phasing the inverted peak so that it is purely emmisive, then adjust the phase of the furthest NOE peak so that it is purely absorptive. The weighting page can be used to increase the line-broadening of the spectrum. This can reduce the amplitude of SPT artifacts as well as improving the SNR of the NOE peaks, but the trade-off is lower resolution. Traditionally, the NOE spectrum is integrated with the inverted peak normalized to 100. However, the NOESY1D is a transient NOE technique and the observed enhancements are different from the older steady-state NOE technique. Furthermore, relaxation during the mixing time prevents simple quantification of enhancement factors. Thus, enhancement values are no longer reported in the literature; rather, qualitative comparisons are used. 5 It s generally best to normalize integrals off of an aliphatic methine or a methylene signal because methyl groups, olefinic hydrogens, and aromatic hydrogens have significantly different relaxation rates. 20 BYU NMR Facility

21 Intro to 1D & 2D NMR PROCESSING & PRINTING YOUR DATA D Specific Processing The vertical scale is changed using the red and blue colored contour level buttons, fig. 10A. Middle-click and the scroll-wheel also change the vertical scale. Decreasing the vertical scale improves the resolution and hides noise, but may also hide short peaks of interest. Increasing the vertical scale will reveal shorter peaks, but may also reveal noise and undesired artifacts. If you have many false cross-peaks or excessive ringing parallel to the F1 axis, you need to re-acquire your spectrum with more t1 increments. If you have excessive noise parallel to the F1 axis or other artifacts, you need to re-acquire your spectrum with more scans per t1 increment. Ringing artifacts in your spectrum can be reduced using the tools in the process tab, weighting page. Click on the gaussian button under the F1 only heading and then press the autoprocess button to apply this weighting to your spectrum. Manually entering a smaller gaussian parameter will further reduce the ringing artifacts, but will also broaden the peaks in F Printing Print your spectrum using the plot page of the process tab, fig. 11. Autoplot, fig. 11A, prints the spectrum using the options set in the drop-down menus. The other plot buttons are difficult to use correctly; stick with the drop-down menus and autoplot/autoplot preview. Both 1D & 2D Parameters, fig. 11C, sets the parameter display style. None turns off parameter printing. 1D only Integrals 6, fig. 11D, sets the type of integral values and where to plot them. Choose scaled integrals if you have normalized a single peak in your spectrum. Use normalized integrals if you normalized the total integrated area of your spectrum. Peak frequencies, fig. 11E, sets where and how to print the peak frequencies. 7 The show buttons beneath the integrals and peak frequencies menus display the values on the screen. You may need to adjust the vertical position of the spectrum in order to see the integral values, sec D only Side-spectra, fig. 11F, are automatically included if you have acquired the necessary 1D spectra. If you have not acquired the 1D spectra, a projection will be used instead. This pseudo-1d spectrum is typically of poor quality and can be turned off by setting the side-spectrum menu to none. 6 The display must be in partial or full integration mode to activate the integrals menu. 7 The threshold must be set correctly first. BYU NMR Facility 21

22 PROCESSING & PRINTING YOUR DATA Intro to 1D & 2D NMR If the default size of the 2D spectrum is smaller than you prefer, use the full or full w/projections button, fig. 11G, and then set 2D plot size to as displayed, fig. 11H. Auto plot preview, fig. 11B, generates and displays a PDF file identical to the output of automatic plot page. Autoplot preview also opens the plot view panel. This panel allows you to take several actions after assessing the PDF preview: Plotter sends the spectrum to the printer (this is equivalent to automatic plot page). File saves the PDF in the plots folder within the study directory. is supposed to the PDF to the address entered in the text box. However, this option is not currently working properly. (Optional) Print your spectrum without a preview using automatic plot page. Figure 11: Processing tab, Plot page. A) print the spectrum using the current options, B) generate a PDF version to preview the spectrum, C) style of parameter reporting, D) style of integral region printing, E) style of peak list printing, F) side-spectra options for 2D spectra, G) size and position of the 2D spectrum, H) auto-resize option for 2D spectra. 22 BYU NMR Facility

23 Intro to 1D & 2D NMR LIST OF FIGURES List of Tables 1 Quickref: Recommended Minimum Concentrations Quickref: Recommended number of t1 increments for 1 H 1 H experiments. 4 3 Quickref: Recommended number of t1 increments for 13 C 1 H experiments. 4 4 Quickref: VnmrJ commands and locations Recommended Minimum Concentrations Recommended number of t1 increments for 1 H 1 H experiments Recommended number of t1 increments for 13 C 1 H experiments List of Figures 1 Linux desktop View menu VnmrJ interface Sample handling Start tab, standard page Study queue and experiment panels Acquire tab, default page NOESY1D setup and processing File browser Processing tools Processing tab, plot page BYU NMR Facility 23