Figure 1: Common Desktop Environment (CDE) has a menu bar at the bottom of the screen. The VNMR icon will open the VNMR software.
Clicking on [GLIDE] on the top menu bar of VNMR will start the GLIDE program. The Custom button and the Go buttons have grey dots on them since they are not setup to run an experiment yet. Select the Setup button to start a new experiment.
Figure 2 : The Glide Menu can be re-sized at the corners and moved around the screen by holding down the ALT key and the left mouse button on the edge of the Glide menu.
On the Setup screen, you can eject and insert your sample. Place your sample in the spinner and measure it with the depth gauge. Remove the dummy D2O sample from the machine and insert your sample. The small down arrows indicate a pulldown menu in VNMR. Holding down the right mouse button on the arrow will open the menu. Use the pulldown menus to select the experiment and the solvent. Typically you will select Proton 1-D for the first experiment. If this is the first experiment you have started on this sample, select Autolock "yes" and Autoshim "yes". On the same set-up screen, there is a line for "Save As:". Click the left mouse button on that line and type in a name for the sample you are running. In the text box below, you can enter any text you want to help you identify the sample. (i.e. information on solvent, pH, concentration, date etc.) After you are finished, click on (Setup). The computer will then set up the pulse sequence. When it is finished, a Custom pulldown menu should appear.
Figure 3 : The Custom pull down menu appears after setting up an experiment and can be re-opened after an acquisition by clicking on the arrow down on
the Custom button.
Click on Acquire on the pulldown menu. The acquire menu will allow you to customize some of the parameters for the experiment. For a proton 1-D experiment, you may select a spectral window in ppm and the number of scans. Be sure to use a wide enough ppm range to encompass every signal you expect. The relaxation delay may need to be longer if you have a weak sample of a small molecule that may have a long relaxation time. The proton pulse angle may also be chosen. A shorter angle allows you to run a shorter relaxation delay and more scans in less time. A typical value is a 45 deg. angle. When you are finished, click on (Do). The instrument will then begin acquiring your experiment.
Figure 4 : The standard 1-D proton options include specifying a
spectral window (in ppm), the number of proton scans, a relaxation delay,
and the option of a specific pulse angle.
The instrument will first do the deuterium gradient autoshimming on your sample. The lock and the spinner should be off in the acquisition status window. If you are running with a CDCL3 solvent, the shimming may take a little longer. After shimming, the spinner will turn on and the instrument will autolock your sample and then acquire the spectrum. The display screen will say "Done" when the acquisition is finished. The acquisition status window will report the Remaining Time so you will know when your spectrum will be done. If the pulldown menu from the Custom Button (Figure 3) is not visible, click on the down arrow of the Custom window to open it. Click on Process to transform your data. The Process button gives you the option of setting a fourier number and a line broadening function. You may select 'No' on both and not use these options. The fourier number will set a zero filling number if you use it. For example if you take 16000 points (np), you could set the fourier number to 32000 and zero fill out to 32000 points. Zero filling can make your spectrum look a little less noisy. The line broadening is a weighting function that is applied to your FID. The smaller the line broadening number, the more resolution is enhanced but some signal intensity may be lost. A larger line broadening (i.e. 1 or higher) will enhance signal intensity but your resolution may reduced. Be sure to choose "Set" to apply the line broadening or zero-filling. Click on (Process). The spectrum should appear on the screen. To make a plot, select the Plot button from the Custom pulldown menu. The Plot buttom provides options for your plot including plotting parameters, integrals and peak picking.
Figure 5: When processing a 1-D spectrum, you have the option of setting
a fourier number and a weighting function. Selecting 'No' turns both off.
If you accidentally exit the Glide window before you have processed and plotted
your data, the Custom button may not be accessible if you re-open Glide.
One option you have at this point is to click on [Main Menu] on the menu
bar and choose [AutoProcess] and then [AutoPlot].
When you finish running experiments:
1. Remove your sample and replace it with the D2O sample through the Setup
menu of Glide. Then click on (Close) on the Setup menu.
2. Exit Glide.
3. At the command line, type >autolock
4. Wait until the instrument tells you the experiment is finished and the
acquisition status says "Idle".
5. Then exit the software by typing >exit.
6. Then logout of the machine by clicking on the [Exit] in the CDE menu bar.
Click 'OK' in the window that comes up.
Combination Experiments
Many of the most common experiments can be queued together from Glide.
When doing the Setup, choose H1 and H1 Detected as your experiment choice.
When you select "Acquire" from the Custom Pulldown menu, you will have many
choices for different experiments (you may need to use the arrows on the
right side to arrow down).
For example, you set the options for your 1-D proton, then select [gCOSY].
This is a gradient COSY. When you click on this option, another window
will open where you can select the number of transients and the number of
increments for the experiment. The minimum for this is one transient and
128 increments. The minimum settings result in a COSY experiment that will
take about 3 minutes total to run. If you have a less concentrated sample,
run more transients.
After you finish selecting options for the gradient COSY, you should see on the
text window, "Experiment 1: Proton, Experiment 2: gCOSY". The computer is
setting up the experiments in the order you select them.
You can then choose gHMQC and another options window will open. The minimum
on gHMQC in transients/increments will result in an experiment that will
take about 10 minutes to run. If your sample is not concentrated, run more
transients. Be sure to select a carbon sweep width that will include any
carbons with a proton attached. After you finish selecting gHMQC options,
the text screen should show the gHMQC as experiment 3.
In this manner you may also add other experiments like NOESY, TOCSY, and
gHMBC. All the experiments will be run in order and plots will come out for
each one. All the data will be saved under the one directory name that you
provide in the Setup window.
How do I choose a mixing time for TOCSY or NOESY?
TOCSY mixing time is in milliseconds (ms). A TOCSY can provide information
about an entire spin system. If you set a short mixing time (i.e. possibly 30ms),
you may only get peaks to the nearest neighbors, similar to a COSY. If you want
the entire spin system, select a longer mix time (i.e. possibly 80 ms).
Sometimes more than one TOCSY experiment is run at different mixing times to
see the short range neighbors and one at a longer mixing time to see the full
spin system.
If you choose the [Array] option in 1D TOCSY, many different mixing times will
be run and the experiment will take a long time. Selecting one mixing time
at a time is recommended.
NOESY mixing time should be fairly long for small molecules (<1000 MW). For
small molecules, select a mixing time of 500 ms or larger. If you do not
detect NOEs you expect to see, try a longer mixing time. NOEs are also
relatively insensitive compared to TOCSY peaks. Also, try running many
more scans for a 1-D NOESY.
Running a 1-D Selective Experiment: 1-D TOCSY, 1-D NOESY If you have already run an experiment on the current sample, choose 'No' for both the autoshim and autolock during the Setup. Choose 'H1 and H1 Selective' for the Experiment.
The Glide package will run a 1-D proton spectrum first and then stop. The spectrum should appear on the screen and the mouse menu bars in the main VNMR should have a menu line that includes options for [Select] and [Proceed]. Using the cursor, click the left mouse button on the left side of the peak you wish to irradiate and click the right mouse button to the right of the peak. Try to get as close to the peak as you can without cutting into it. Then choose [Select].
If you wish to do 1-D selective experiment on more than one peak, choose the second peak as you did the first and choose [Select]. After you are finished selecting peaks, click on [Proceed]. The 1-D selective experiment(s) will then be acquired. To get the ppm scale to appear on a print out from a 1-D TOCSY or a 1-D NOESY: At the command line type >pscale and then hit the [Autoplot] button. 1-D TOCSY acquires two separate spectra: one with the peak irradiated alone and one with the TOCSY peaks included. The irradiated peak should be positive. To see the second spectrum only, use >ds(2) 1-D NOESY acquires one spectrum with the irradiated peak negative and the NOE peaks positive.
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