NMR INSTRUMENT INSTRUCTIONS: Safety and Sample Preparation

Transcription

1 NMR INSTRUMENT INSTRUCTIONS: Safety and Sample Preparation The GVSU chemistry department owns 2 NMR spectrometers. A JEOL Eclipse 300 MHz and a Varian Inova 400 MHz. Due to their strong, constant magnetic fields NMR instruments have specific safety concerns. Please read the short safety section below. NMR Safety NMRs use strong magnetic fields. If you depend on a medical device that is sensitive to magnetic fields Do Not Enter the NMR Room. Do not approach within 5 feet of the magnet if you have iron or steel objects. Do not bring cell phones and magnetic media such as credit cards near the magnet; leave them in the lab or near the instrument computer. Sample Preparation is identical for either instrument but varies depending upon which nuclei you are investigating. NMR active isotopes are naturally present in different abundancies. The 100% abundance for 1 H nuclei allows for more dilute samples than 13 C, naturally present at 1.1% of carbon atoms. Preparing a HNMR sample For most HNMR samples you will want a 0.1M solution of your sample dissolved in a deuterated solvent often CDCl 3 (Chloroform D). This concentration corresponds to between 5 mg and 25 mg or 1-5 drops of sample in 0.7ml of deuterated solvent. Filter the solution if necessary to remove solids and transfer the solution to a 5mm NMR tube. The liquid level should be about 3 fingers or 50mm high in the NMR tube. Solvent height is important for obtaining a good spectrum. Preparing a CNMR sample Due to the lower abundance of C-13 isotope your solution should be more concentrated than a typical HNMR sample. For most purposes you will want a 0.5M solution (25 mg or 5-10 drops) of the sample of interest dissolved in 0.7ml of deuterated solvent - CDCl 3 - and transferred to a 5mm NMR tube. 13C is much less sensitive than 1H. Either make the sample concentrated or plan on more scans. Common steps used to obtain spectra are outlined below. More specific directions for use of each instrument follow. Steps for Obtaining NMR Spectra 1. Prepare a sample 2. Login to NMR computer and connect to spectrometer operating system 3. Insert your sample and lock on sample 4. Run the experiment 5. Process your spectrum 6. Eject your sample and reinsert instrument standard 7. Lock on the standard 8. Clean your NMR tube

2 INSTRUCTIONS FOR OBTAINING A JEOL 1H NMR SPECTRUM A. Sample preparation: Before you use the NMR 1. Prepare a sample as directed on page The NMR tube is lowered into the machine using a tube holder called a spinner. A graduated depth gauge will help you determine how deep to place the tube in the spinner. Put an empty spinner in the depth gauge. Push the center portion of the spinner down all the way; this is crucial to machine operation. Now, gently insert the NMR tube into the spinner, pushing the tube to the bottom of the depth gauge. Holding the spinner by its wider upper part remove spinner and tube from the gauge and clean with a Kimwipe. Be careful not to change the depth of the tube in the spinner as you clean. Recheck the tube depth before putting it in the auto sampler. The autosampler is an 8 tube arrangement at the top of the NMR. Place the spinner and tube in one of the empty slots in the side of auto sampler nearest the computer. Never place the spinner into the slot that is over the NMR probe (back of auto sampler)! It will just fall down on top of the sample currently there! Figure 1. JEOL Spinner, NMR tube and Depth Gauge Depth Gauge Spinner B. Connecting the NMR computer and your sample The JEOL NMR uses a program called Delta to run and process samples. Delta is UNIX based software. To make entries in Delta the computer cursor needs to be in the field where you are adding information. NMR tube 1. Computer Login and connection to NMR software Click on the appropriate login icon: See your research advisor/class instructor If you do not see an array of login icons go to switch user in the Start menu. Double click on the Delta icon on the desktop. This opens the Delta console. In Delta, select File-> Preferences. Under the Directory/Folder tab check that your data will be stored in the desired folder. Within the Delta console window click on the icon for the spectrometer control. This is the fourth icon in the second row and is circled in figure. A window for spectrometer control Figure 2. Delta window will open. You will likely see a line with the text "eclipse2 - FREE "

3 Within a brief moment the two computers will connect. In the yellow bar you should see Connect: eclipse2jeol.com". If it does not immediately connect: you may select the eclipse line (by clicking on it) and click on the yellow Connect button. Figure 3. Spectrometer Control 2. Locking the Sample. This procedure helps maintain a consistent magnetic field during the NMR experiment. In the Spectrometer control window Click on the button labeled Sample (lower left corner). The Sample window will open - see the figure 4 below. Under Sample State choose the auto sampler slot where your sample is by clicking on the arrows. Once you move your cursor out of this window the auto sampler will eject the current sample and lower your sample into the probe. Under Solvent select the deuterated solvent you used. Under Lock Control chose gradient shim and lock - the third icon to the right, 2nd row. Wait until the instrument completes the gradient shim and lock experiment before continuing. This will take some time. Figure 4. Sample Window Sample slot Solvent Lock Status

4 C. Acquiring a Proton NMR Spectrum Figure 5. Lower Spectrometer Control window 1. Setting up an Experiment Once you have completed locking on your sample you may close the Sample window and return to the Spectrometer control window, figure 5. Select the Experiment box at the bottom of the window (shown circled). You will Figure 6. Open Experiment window see the Open Experiment window. 2. In the Open Experiment window set the type of experiment. If you do not see a list of files under Filename, you will need to click on the global folder: In the global folder scroll down under Filename to the file: single_pulse.exp Select this file and click on ok. The Experiment Tools window opens: 3. Within the Experiment Tools you will need to set some parameters, though most are set adequately by default. You should be in the Header tab. Put the Figure 7. Experiment Tools window cursor in the filename field and enter a filename. Your spectral data is stored in a folder based on your username and this filename. A good filename choice is your initials, followed by a brief experimental descriptor, eg: CM_s01_exp2_1HNMR When looking for this file remember UNIX is case sensitive. Enter additional info in the sample id field to convey more information about the sample or experiment. Click the auto_gain box Entries in other tabs are generally not needed. Click on Submit in the upper right of the window to start your experiment. The experiment is entered into the NMR s queue and will be run on a first-in, first-out basis. A pop up will appear and ask you to acknowledge addition of the experiment to the queue. Accept this by clicking Go. You can see the experiment queue in the Spectrometer Control window. In this window you may abort an experiment by clicking on the experiment in the queue and clicking on the Stop button. When the experiment is complete a data processing window will open with the spectrum.

5 D. Processing a Proton NMR Spectrum After measurement is complete, data is automatically forwarded from the spectrometer, and processing is automatically executed (If this is not done click on the 1H button and then click on the Process button). Figure 8. The 1D Processor window This section explains data processing in the 1-D Processor. The window usually displays two forms of the data. The upper display is the Free Induction Decay (FID) which is raw data from the spectrometer (display of the FID can be unselected in the preferences menu). The lower display is a more familiar spectrum processed by Fourier transformation (FT). Processing cursor tool On most spectra you will want to set a reference peak, peak pick important peaks, integrate the peaks and measure J-couplings. Short descriptions of these procedures are given here. The Delta manual has much more detail on processing. 1. Set a Reference Peak in the Display menu. Choose Display > Reference > Automatic Reference to set the reference. You will need to unselect the TMS radio button. Click on Process in the upper right. 2. Automatic integration and peak picking are obtained by clicking on the icon with an integral sign and octagon in the icon bar of the 1D Processor window. Figure 9. 1D Processor window icons 3. Determine coupling. The processor tab button in the upper right corner of the spectrum opens a series of processing options (see figure 8 of processor window above). In Peak mode of the cursor tool click on more peaks tool. On your spectrum select the 2 peaks where you want to measure a J value. A hashtag appears above the selected peak. Type "j" to assign a value. Type "u" to unselect those two peaks, repeat as necessary. More detailed information on processing spectra can be found in the Delta manual.

6 E. Finishing up 1. Eject your sample and return the standard to the NMR Click on the Sample button in lower left corner of Spectrometer Control window to go back to the Sample window. You need to retrieve your sample and replace it with the standard sample that was in the NMR. This is similar to loading your initial sample. In the Sample window place the cursor over the sample slot box Input or click arrows to indicate slot 1, the value for the slot holding the standard sample. Move the cursor out of the box. The instrument automatically ejects the current sample and loads the standard Figure 10. Sample window that is in auto sampler slot Lock on the standard sample in slot 1. Select the Solvent Sample usually it will be D2O slot Click on the icon to load system shims. Click on the icon for locking the sample. Close the sample window. 3. Disconnect NMR and Logoff In Spectrometer Control window click on the Unlink button. You are no longer in control of the NMR. Close the spectrometer control window. Record your experiment in the NMR log. Make note of any problems that occurred Log off the computer. Congratulations you have acquired an NMR spectrum. Lab Master IP address: Remove your sample and Clean your NMR tube After logoff retrieve your sample from the auto sampler and take it out of the spinner. Place the spinner back in foam spinner rack. Pour your NMR sample in the halogenated waste in the hood. Place your empty tube on the tube cleaner and rinse with acetone. Save the tube for reuse.