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USING THE NMR Sample Preparation for NMR In order to get

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									USING THE NMR


Sample Preparation for 1H-NMR
In order to get a spectrum of good quality you need to prepare the sample properly. The
following are rules for preparing samples:
        - You need to use only about 0.7mL of dueterated solvent (this is about 4cm)
        - No solids should be present in the NMR tube. Filter your sample if you need
            to using a Pasteur pipet with a plug of glass wool or cotton in it.
        - Use Wilmad 507-pp NMR tubes
One of the most common questions is “how concentrated does my sample need to be?”
Well, the more concentrated a sample, the lower the amount of time it will talk to collect
a good spectrum. In general for 1H samples you should have a concentration of 5% or
0.3M. You can use a less concentrated sample, it will just take more time (maybe 2-3
more minutes).

**YOU CANNOT JUST PUT YOUR SAMPLE IN THE NMR TUBE INTO THE
NMR**

You need to put the NMR tube into a sample spinner (see figure 1)



      NMR tube
      with cap                                     NMR tube in spinner




                                                 Figure 1. NMR tubes and spinner




Make sure you insert the NMR tube into the spinner in the correct orientation! The wider
part should be closer to the top of the NMR tube! Now, how do you know how far into
the depth gauge the NMR tube needs to be? That is what the depth gauge is for (figure
2).
                                               Figure 2. NMR tube in the spinner
                                               placed in the depth gauge




Gently place the NMR tube with the spinner into the depth gauge hole. The NMR tube
should be touching the bottom of the gauge (press the top of the tube gently) and the
bottom of the spinner should be touching the top of the depth gauge opening.

Help! I can’t find the Depth Gauge hole!

Below is a picture of the entire instrument showing where the important components are.




                                                (c) Lid for
                                                sample                   (d) Depth
                                                insertion                Gauge
                                                                         Hole




                                                              (e) NMR
                                                              Magnet




    (a) Airflow              (b) Computer
    on/off                   on/off

     Figure 3. Anasazi NMR
Collecting 1H-NMR data

Now that our sample is prepared and placed at the correct depth into the spinner, you
need to put it into the NMR to collect some data.
Turning things on
       - Make sure the computer (Figure 3 (b)) and the airflow (Figure 3(a)) are turned
           on!
       - Two computer programs will automatically open, WinPNMR and NUTS.
           WinPNMR allows for the collection of data while NUTS processes the data
           you collect. Make sure you are in the WinPNMR window (see figure 4)




              Figure 4. WinPNMR window

Inserting your sample into the instrument
On the top of the NMR magnet is a cover plate. This is where your sample will go.
Figure 5 shows what it looks like with the cover plate open.




Figure 5. Sample insertion chamber containing (from top to bottom) the hole for the
sample (probe), a toggle switch, and an airflow button.
       -   Before you insert your sample, make sure the airflow is on!
       -   Press the airflow button (see fig 5) to remove the sample that is already inside
           (and to get the spinner too!)
       -   Insert your sample into the “Probe” hole. It will sink a bit, and then float.
       -   Move the toggle switch towards the front of the instrument to drop the sample
           into the probe.
       -   Press the toggle switch back to turn on the spinner. You should look down
           into the hole to make sure that it is spinning!

Shimming the Magnet
We shim the magnet to optimize the resolution of the NMR (so that our spectrum looks
nice). At this point your sample should be in the NMR!
       - Make sure you are in the PNMR window.
       - Look at the prompt in the bottom left hand corner of the window and make
           sure it says “H1>”. This means that we are taking a proton NMR. If is says
           something else, type nu H1 and then press <enter>
       - Type shim and then press <enter>
       - A dialog window will appear asking you to enter in an RD value. If you are
           running a dilute sample type 5 or if you are running a neat sample type 2
       - A Pulse Program Output window (gray) will appear and the dialog inside of it
           will change as the program progresses
       - Now you have to wait. When it is done the gray Pulse Program Output
           window will disappear.

Setting a reference peak
Now we need to make sure that our peaks are appearing where we think they should be.
We do this by using an internal standard. This standard has a known chemical shift that
we can calibrate the scale with. Often TMS is used as an internal standard, but you can
also use the NMR solvent as the standard (ie CDCl3 or D2O). Regardless, the instructions
for setting a reference peak are the same except for the value you will enter in.
         - Make sure you are in the PNMR window
         - At the H1> prompt, type zg
         - Press <enter> <enter> to accept the default file for saving the data
         - On the screen you should see an FID (free induction decay), looking
            something like that in figure 6.
Figure 6. FID – this one is yellow, which is good

       -   A gray dialog box (“Running zg”) will appear in the upper right hand corner
           of the screen.
       -   When data collection is complete, the gray box will disappear. If the FID is
           yellow you can proceed directly to the next step. If it is RED, then you need
           to decrease the gain. (ask the instructor)
       -   Now we need to see the spectrum. This is done by converting the FID into the
           peaks that you are used to seeing.
       -   Enter the NUTS window by pressing <Alt> + <Tab> simultaneously (or
           clicking on the task bar at the bottom of the screen)
       -   Type a2 and then press <enter>, this should bring up a picture of your
           spectrum in the window.
       -   Place the cursor on the reference peak. This can be either TMS if it was
           added, or the solvent. Note the reference peak position in ppm, including
           sign.
       -   Switch back to the PNMR window by pressing <Alt> + <Tab>
           simultaneously. At the H1> prompt type fo and press <enter>
       -   A dialog box will appear. Type in the current value for your reference peak,
           and then the standard value for that reference peak. A list of standard values
           is given below (also see the Cambridge Isotope Labs sheet attached):
               o TMS – 0 ppm
               o CDCl3 – 7.24 ppm
               o D2O – 4.81 ppm
       -   **every time you switch to a different solvent you must re-shim (start at the
           beginning of this section**

Collecting a 1H-NMR
You are now ready to collect your sample spectrum. Your sample is already in the NMR
so now we just need to collect the data.
       - Make sure you are in the PNMR window
       - At the H1> prompt type zg and press <enter>
       - Press <enter> <enter> to accept the default file for saving the data
       -   On the screen you should see an FID (free induction decay), looking
           something like that in figure 6.
       -   A gray dialog box (“Running zg”) will appear in the upper right hand corner
           of the screen.
       -   When data collection is complete, the gray box will disappear.
       -   Press the <alt> + <tab> buttons simultaneously to go back to the NUTS
           program.
       -   Press <Ctrl> + <F2> simultaneously, and then click “open”. This will bring
           up a window where you can type your name, the date, and the sample number.
           Once you have entered in this information, click “ok”. Your spectrum should
           now appear on the screen.

Data analysis
Now that you have your NMR spectrum you need to phase it, integrate and label the
peaks!
       - Your spectrum should consist of one or more peaks. If you see no peaks other
          than your solvent peak, go back to the beginning of “Collecting a 1H NMR”
          and change the number of scans that you are doing. You can do this by typing
          ns at the prompt in PNMR and then when the dialog box opens, type in 12. If
          you still don’t see any peaks, call the instructor.
       - These peaks should be surrounded by a flat baseline.
       - Now we need to phase the spectrum (if some of the peaks dip below the
          baseline)
              o Type zo to set us up for phasing
              o Click, hold and drag the cursor over a large peak(s) on the left of the
                  spectrum, the release, and press <1> (number one)
              o Click, hold and drag the cursor over a large peak(s) on the right of the
                  spectrum, then release and press <2> (number two)
              o Press <enter>
              o At the prompt type pe
              o Phase the left side peak by pressing and holding the left mouse button
                  while dragging the mouse left or right until the peak looks symmetrical
                  and is not below the baseline. Repeat for the right side peak by
                  pressing and holding the right mouse button.
              o When you are done, press <enter>
       - Type fb (in NUTS) and any areas that are baseline (flat) should be colored
          pink. If there are flat areas that are not pink, click the mouse over that area to
          make them pink.
       - Type l (letter L) and hit <enter>
       - In order to label the chemical shift of the peak in ppm, type pp at the prompt.
       - Type id at the prompt. This will display the integral for each peak. Type c to
          obtain a continuous integral line. Using the mouse, place the cursor on the left
          side of the farthest left peak and click the left mouse button twice. Place the
          cursor on the right side of the peak and click the left mouse button once. This
          will select that peak to be integrated. Repeat the procedure for all peaks in
          your spectrum except for your solvent and TMS (if present).
       -   In order to display accurate values for the integrals, you need to find a peak
           where you think you know the number of hydrogens, place the cursor on top
           of that integral, left click and type the letter v. Under “Current relative value”
           type in the number of H’s you think that peak represents. The rest of the
           integrations will display values relative to this one. If you guessed wrong or
           think that your integration is incorrect, you may change it by repeating the
           above for any of the peaks on the spectrum. When you are done, press
           <enter>. You can turn on and off the integrations by pressing <Ctrl> + <I>
           simultaneously.
       -   FINALLY, we can print our spectrum! At the prompt type pl

To finish!
Now you must remove your sample from the NMR, put back in the tube that was in there
to begin with, and turn everything off.
        - Press the airflow button (fig 5) and up comes your sample
        - Put the water standard (yellow cap) into the spinner to the correct depth (use
           the depth gauge!)
        - Insert the water standard into the “Probe” hole (with the spinner). It will sink
           a bit, and then float.
        - Move the toggle switch towards the front of the instrument to drop the sample
           into the probe.
        - Press the toggle switch back to turn on the spinner. You should look down
           into the hole to make sure that it is spinning!
        - Turn off the computer (Start -> shutdown)
        - Turn off the airflow
        -

								
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