IMSERC NMR MANUAL 05: Manual Operation of Agilent NMR Spectrometers (Chem350 Interface)

Transcription

1 IMSERC NMR MANUAL 05: Manual Operation of Agilent NMR Spectrometers (Chem350 Interface) Last updated: October 12, 2011 by Josh Kurutz THIS PAGE = QUICK START GUIDE 0) At the computer, make sure VNMRJ is running under the account named northrup, and that the instrument is locked & not acquiring data 1) Load a new PROTON experiment by pulling down Experiments Proton from the main menu bar 2) Eject the CDCl 3 ( lock ) standard sample from the Start/Standard panel by clicking Eject 3) Remove the sample from the magnet 4) Carefully load your sample into a spinner using the sample positioner 5) Place your sample on top of the magnet so it floats on the (loud) eject air. On Au400, you will need to push it past the silicone grabbers, holding the spinner, not the sample tube. 6) Insert your sample by clicking Insert on the Start/Standard panel 7) Select your solvent in Start/Standard pulldown menu 8) Retrieve the standard shims by clicking the light blue retrieve default shims button. 9) Lock on the sample with the manual locking procedure in the Start/Lock tab. 10) Shim on the sample using the manual shimming procedure. 11) Make sure your sample is locked and that the lock level is between ~15 and ) Move to the Acquire/Default H1 panel by clicking on the Acquire panel s tab and then the Default panel title. 13) Set the number of scans to 1 and acquire a preliminary 1D 1 H spectrum by clicking the green Acquire button. 14) Evaluate the spectrum examine spectral width, phasing, linewidth, referencing, etc., and make necessary changes. 15) If necessary, acquire more scans; in the Acquire/Default H1 panel, pull down the number of scans pulldown to the desired number of scans a. NB: Keep in mind that S/N improves with the square root of the number of scans; increasing them from 4 to 8 will increase S/N by a factor of ~1.4, but from 4 to 16 will improve it by a factor of 2. 16) Save your spectrum by clicking of the floppy disk save button 17) Continue with further experiments and other samples if you have them 18) When you are done with your last sample, eject it using the Eject button 19) Insert the lock standard using the Insert button in the Start/Standard panel 20) Sign out from the pen-and-paper logbook, noting any instrument problems 21) Process your spectrum further and perhaps print it out using an offline processing computer 1

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3 Manual Operation of Agilent NMR Spectrometers (Chem350 Interface) Setup and Acquisition of a 1D 1 H NMR spectrum Step 0) At the computer, make sure VNMRJ is running, and that the instrument is locked & not acquiring data If necessary, wake up the computer by moving the mouse or typing a key on the keyboard (preferably the spacebar). If you are unfamiliar with the layout of the VNMRJ window, you should take a minute to orient yourself: Figure 1. Orientation to the VnmrJ window First, navigate to the Start/Standard panel, indicated in the lower half of the VNMRJ window. 1

4 Toward the upper left corner of the Start/Standard panel, locate the Operator designation. For Chem350, it should read northrup. To determine whether the instrument is locked, look in the lower-left corner of the VNMRJ panel (near the trash can) and read the number under the word Lock. If the number is blue and above ~15, the system is locked. Figure 2. Operator designation & lock status * PROBLEM? If the instrument is not locked on the lock standard when you walk up to the instrument, either the lock circuit is not engaged, there is no sample in the magnet, or the shims are too poor to support locking. Proceed normally, but when you try to eject the sample that should be in the magnet, make sure that a sample emerges. If no sample comes out, contact staff immediately. Usually when you walk up to the instrument, the VNMRJ program is running. If it is not, you ll just see the underlying Linux desktop for the login account northrup (Figure 3): Figure 3. Essential parts of the linux desktop If VNMRJ is not running, start it by clicking on the VNMRJ icon on the Linux desktop (Figure 3). 2

5 Step 1) Load a new PROTON experiment by pulling down Experiments Proton from the VNMRJ main menu. You can see that the spectrometer offers a large number of experiments, but the Proton experiment is the choice on top. Figure 4. Start a new 1H 1D experiment using fresh parameters by pulling down the VnmrJ Manin Menu's pulldown labeled "Experiments" to the "proton" choice. 3

6 After selecting the Proton experiment, your main display window should show you a diagram of the pulse sequence for the PROTON spectrum. Make sure you are viewing the correct panel in the lower part of the display; click on the Start tab and the Standard panel label. Figure 5. The Proton pulse sequence and parameter interface. Step 2) Eject the sample from the Start/Standard panel by clicking Eject Figure 6. The eject button You should see the lock standard floating on top of the magnet. The lock standard is simply a sample of CDCl 3 in a flamed-sealed tube with a red-painted top. IF NOTHING COMES OUT OF THE MAGNET WHEN YOU CLICK EJECT, GET HELP FROM FACILITY STAFF IMMEDIATELY. 4 Figure 7. The Lock Standard floating atop the magnet

7 Step 3) Remove the lock standard from the magnet. Be careful to lift it all the way out before bringing it toward you. The most common way for people to break samples in the magnet it to snap off the tube below the spinner as they remove the sample from the magnet. Figure 8. Carefully removing the sample from atop the magnet. IF YOU EVER BREAK A SAMPLE IN OR NEAR THE MAGNET, YOU MUST ALERT IMSERC STAFF RIGHT AWAY! IF YOU BREAK A SAMPLE IN THE MAGNET BETWEEN 8 AM AND 10 PM, YOU MUST IMMEDIATELY CONTACT JOSH OR YUYANG IN PERSON OR BY PHONE AND AND PLACE A STOP SIGN FROM (obtained from the front of the lab, near the login computers) ON THE KEYBOARD! BEWTWEEN 10 PM AND 8 AM, YOU MUST PLACE A STOP SIGN ON THE INSTRUMENTʼS KEYBOARD AND JOSH AND YUYANG AND SUBMIT A PROBLEM REPORT WHEN LOGGING OUT OF THE FOM. YOU WILL NOT BE CHARGED OR PENALIZED FOR BREAKING A SAMPLE!!! SAMPLE BREAKAGE DOES NOT NECESSARILY CAUSE MUCH DAMAGE TO THE INSTRUMENT, BUT IF THE NEXT SAMPLE IS INSERTED OVER BROKEN GLASS, IT WILL BREAK THE PROBE, CAUSING THOUSANDS OF DOLLARS WORTH OF DAMAGE AND POTENTIALLY SEVERAL WEEKS OF INSTRUMENT DOWNTIME. 5

8 Step 4) Carefully load your sample into a spinner using the sample positioner Figure 9. Position the sample in the spinner. Step 5) Step 6) Place your sample on top of the magnet so it floats on the (loud) eject air. On Au400, you may need to push it past the silicone grabbers; simply hold the spinner not the probe and push it past the grabbers. Insert your sample by clicking Insert on the Start/Standard panel Figure 10. The Insert button. 6

9 Step 7) Select your solvent in Start/Standard pulldown menu Figure 11. Selecting the sample's solvent. Step 8) Retrieve the standard shims by clicking the light blue read default shims button in the Start/Standard panel. Figure 12. Reading the default shims 7

10 Step 9) Lock on the sample with the manual locking procedure in the Start/Lock panel. First, navigate to the Start/Lock panel. Second, turn the Lock off by clicking the OFF button in the lock panel. Third, begin observing your 2 H lock signal by clicking the Lock Scan button: Figure 13. The Lock panel The signal you see is the first part of the 2 H FID. Any waves you observe arise from the beat frequency difference between the fixed spectrometer frequency and the current resonance frequency of your solvent deuterium atoms. Your goal is to adjust the resonance frequency of your solvent so that it matches the transmitter frequency of the spectrometer s lock channel. You make those adjustments by changing the magnetic field strength, which you accomplish by changing the strength of a homogeneous corrective field called Z0 named because its field is the same across the length of the sample, thus has a zeroth-order dependence on Z. Now adjust the magnetic field by changing the value of Z0. Large-scale changes can be effected using the slider bar, but they usually aren t necessary. Left-click to make Z0 lower, right-click to make it higher, and center-click to toggle through the amounts Z0 will change (±1 ±10 ±100 ±1 ). You generally just pick a direction, change Z0, then observe whether the number of beats in your signal increases or decreases. If it decreases, continue changing Z0 in that direction; if it increases, turn around because you re going the wrong way. Figure 14. Making Z0 adjustments 8

11 You should see changes in your 2 H lock signal: Figure 15. Lock signal when Z0 is incorrectly set If you make adjustments to Z0 and do not observe any signals, you may need to increase your signal strength. Do this first by amplifying the lock signal more by increasing the Lock Gain; accomplish this by right-clicking on the Lock gain button. If your signal is too noisy or you ve reached the maximum gain, try applying a stronger lock RF field to increase your 2 H magnetization; increase your lock RF power by right-clicking the Lock power button. Figure 16. Adjustments for lock gain and power You should ultimately get a signal that looks like the one below, or at least one that looks close. When you ve done this, click the On button on the Lock panel to engage the lock feedback loop. The lock status should then read Regulated. Figure 17. G00d-looking lock signal, regulated. 9

12 Step 10) Shim on the sample using the manual shimming procedure. First, navigate to the Start/Shim panel: Figure 18. The Start/Shim panel. The purpose of shimming is to make the magnetic field across the sample more uniform, but we do not generally assess field homogeneity directly. Instead, we use the lock level to gauge shim quality. In the figure above, the lock level reads 13.5 (on a 0-to-100 scale), and on the screen will probably flicker between 13.3 and To shim we adjust the current running through various non-superconducting shim coils in the magnet. We accomplish this by adjusting the setting of various buttons, primarily Z1 and Z2, using the left-, right-, and center-buttons on the mouse just as was done when adjusting Z0 to establish the lock. Left-click to make each shim lower, rightclick to make it higher, and center-click to toggle through the amounts the shim will change (±1 ±10 ±100 ±1 ). One does not know which way to adjust each shim at first it is an empirical process. For each shim, choose one direction of adjustment and assess whether the lock level rises or falls. If it falls, switch to the other direction. If it rises, continue in that direction until it falls, then back up and stay with the shim value that corresponds to the maximum lock level. Then move on to a different shim. Here we start by adjusting Z1 from to to reach a higher lock level (29.8): Figure 19. Adjusting Z1 improves the lock level. 10

13 Then adjusting Z2 from 1458 to 1428 yielded a further gain in lock level (to 35.2). Figure 20. Adjusting Z2 improves the lock level further. Z2 and Z1 shims are dependent on one another, so after adjusting Z2, go back and adjust Z1. If you want further improvement, you may wish to adjust Z3, X, XZ, Y, and YZ. One can spend all day shimming! To avoid that, you probably want to stop when no large improvements can be made. At this point, you should acquire a spectrum and assess whether the observed lineshape is good enough for your purposes. Step 11) Make sure your sample is locked and that the lock level is between ~15 and 90. If you need to adjust it, first change your lock gain (probably setting it lower), then change your lock power. Below a lock level of 15, you risk losing the lock control. Above that, and you may have trouble with your lock level going above 100 if you improve the shims further. There s no magic lock level number you re aiming for when shimming. Be careful to not increase the lock power too much; you may induce saturation of the 2 H signal, in which the lock level fluctuates or is unreliable. Step 12) Move to the Acquire/Default H1 panel by clicking on the Acquire panel s tab and then the Default panel title. Figure 21. The Acquire/Default H1 panel. Step 13) Set the number of scans to 1 and acquire a preliminary 1D 1 H spectrum by clicking the green Acquire button. Under Acquisition Options, pull down the Number of Scans menu to 1. Then click Acquire. 11 Figure 22. Selecting the number of scans.

14 Step 14) Evaluate the spectrum examine spectral width, phasing, linewidth, referencing, etc Figure 23. Spectrum viewing tools. Step 14a) Examine some of the prominent lines in your spectrum more closely by zooming in on them. Place your left cursor to the left of the peak with your left mouse button : Figure 24. The full 1D 1H spectrum, starting to zoom in. Next, place your right cursor to the right of the peaks by right-clicking : Figure 25. Full 1H 1D spectrum, including the right-cursor for zooming. 12

15 Next, click the zoom in button (magnifying glass with + sign, ): Figure 26. The spectrum, zoomed in upon. If you wish, increase the vertical intensity of the spectrum by center-clicking above a peak. This will bring the point in the spectrum directly underneath your cursor right up to the cursor. You can also use the scroller wheel to adjust intensity. Figure 27. Spectrum portion with vertical scale adjusted. Does that look OK to you? It may be hard to tell unless you ve investigated that spectrum before. It s often better to look at the solvent peak, since that shouldn t change significantly from sample to sample. Step 14b) Start gauging your spectrum quality by examining the linewidth of the residual solvent peak (aka, the solvent peak, but which is really the protiated portion of the deuterated solvent. Remember that 99.9%-deuterated CDCl 3 will still have 0.1% CHCl 3, which you observe and can even use as a chemical shift reference.) This is easier for solvents that present single lines, like CDCl 3, than solvents with multiple lines arising from 2 H- 1 H coupling, like dmso-d6. 13

16 Get a better look at your residual solvent line by zooming in on it; you may need to zoom out to the full spectrum first, which you accomplish using the zoom out button:. Figure 28. Zoom in on the solvent line. Next, you need to place your cursor precisely on the solvent peak. You can accomplish this in two steps: first by placing the (left) cursor somewhere on the peak. Figure 29. Place you cursor on the peak. Then, in the Process/Weighting panel, (1) click find nearest line, then (2) display linewidth. Your linewidth will read out in the display windows. Your solvent line should be below 2.0 Hz for routine data, and ~1.0 or below for publication quality. Figure 30. Find nearest line, display linewidth. 14

17 Step 14c) While your cursor is on the tip of the solvent peak, you may also wish to ensure your spectrum is accurately referenced with respect to frequency in ppm. The most definitive way to reference your spectrum is to include a small amount of tetramethyl silane (TMS) in your sample, and ensure that its 1 H peak resides at ppm; this is the IUPAC-approved method for organic samples. If you have no TMS in your sample, which is very common, you can reference the spectrum to the chemical shift of the residual 1 H signal from the solvent. To confer the correct chemical shift on the solvent peak, leave the cursor on top of the peak, navigate to the Process/Default panel, and under the Reference section locate the Reference cursor to box. In that box, type the correct chemical shift of the solvent; for CHCl 3, you would type 7.24 in that box and hit the return key. This changes the spectrum scale so that the cursor s position reads 7.24 ppm. Figure 31. Manually referencing a spectrum by defining the frequency of a solvent peak. Step 14d) Continue evaluating your spectrum by increasing the vertical scale to see if there are any low-lying peaks that you may wish to observe. If so, you may wish to take another spectrum with a larger number of scans to make these peaks more clear. Step 15) If necessary, acquire more scans; in the Acquire/Default H1 panel, pull down the number of scans pulldown to the desired number of scans a. NB: Keep in mind that S/N improves with the square root of the number of scans; increasing them from 4 to 16 will increase S/N by a factor of ~1.4, but from 4 to 16 will improve it by a factor of 2 15

18 Step 16) Save your spectrum by clicking of the floppy disk save button A dialog window will pop up that will show you the files in the location where the last user saved their spectra. Navigate from here to your own folder by A) Clicking on the folder selector pulldown, which opens the file browser dialog B) Clicking the walkon account s folder C) Clicking (or creating) your folder D) Typing in a legal file name in the File Name box; legal = alphanumerics only, upper/lowercase OK, no spaces or special characters like single or double quotes. Step 17) Continue with further experiments other samples if you have them. Step 18) When you are done with your last sample, eject it using the Eject button in the Start/Standard panel 16

19 Step 19) Insert the lock standard using the Insert button in the Start/Standard panel, and establish lock on it. Step 20) Sign out from the pen-and-paper logbook, noting any instrument problems Step 21) Process your spectrum further and perhaps print it out using an offline processing computer 17