Homonuclear Correlation Spectroscopy
In this lab we will begin to acquire more complex sets of NMR data. Overall the
process to set up the experiments is quite similar to that which we have already
done with the 1D methods with the exception of the presence of a second acquisition
dimension to parametrize. The homonuclear COSY and TOCSY experiments require only
the 1H channel be calibrated for the 90 degree pulse at the default power level.
If time permits these pulses can be recalibrated using a 360 degree determination
centered around 4*the current pw.
To begin we must first have the subject samples locked and shimmed. Once this is
done we will be working with the buttons labeled H1&H1 Detected in the Setup
Exp Tab as shown below:
For those stdents connected to the 300 MHz machine the only option for COSY is a
phase cycled experiment. The following instructions pertain to the phase cycled
experiment on either the 300 or 500 MHz instruments. For those students connected
to the 500 there will be further instructions for doing a gradient selected COSY
Start by clicking on the H1&H1 Detected button which will bring up the now familiar
Saveas and text box after which the setup dialog box pops up as shown below:
In this setup dialog we see the additional choices for doing a COSY experiment as
well as a TOCSY experiment. Other experiment types can also be selected including
the heternuclear correlation experiment (the subject of Lab 3).
The pertinent choices for the COSY experiment include the number of scans PER increment
which defaults to 4. If time permits for the phase cycled experiment it is much
better to complete the full phase cycle of 16 scans. The other choice is for the
number of increments, which has a direct bearing on the resolution of the F1 or
incremented dimension. Again the default is set for time purposes. Note that this
experiment will utilize the d1 (relaxation delay) from the proton experiment, which
again has a bearing on the propensity to have artifacts and "T1" noise
mar the spectrum.
For purposes of fast acquisition the number of scans should be kept to 4 and the
increments left at 64 which results in an 11 minute experiment for the phase cycled
Once the appropriate values have been set click the OK button on the COSY choices
and the OK and Exit buttons on the 1H&1H Detected dialog box. A message will
appear that the 1H spectrum will be run first followed by the COSY.
Click the Acq&Obs tab to begin the experiment with the green Start button. The
proton spectrum will acquire and the COSY experiment will be set up following the
plot of the proton spectrum. The Acq & Obs tab should look like the following:
Note the section for the 2nd dimension in that the acquisition is an "N"
type which means an anti-echo collection via the phase cycle and an absolute value
display up 2D FT.
The data will acquire for the next 11 minutes and you can monitor the process in
the Acqstat window. Once the data is done acquiring the data will be processed,
plotted and the following display will appear:
Reprocess the data by clicking on the Process Tab which brings up the window shown:
Begin by just pressing the Full 2D transform button which will initiate the 2D transform
and produce the following spectrum:
How does this one differ from the one first displayed after the data was collected?
What is the origin of the messy streaks around 2 ppm across the F1 dimension?
To remove the artifacts from the spectrum click on the Display tab to bring up the
window shown here:
By pressing the Symmetrize 2D button the 2D spectrum is returned to the state it
was after the data was first collected. What is the Symmetrizing routine doing to
the data? Is symmetrizing the spectrum always a good idea?
While in the Display mode practice expanding regions of the 2D map in a fashion
analogous to that in the 1D mode. The right mouse button will contol a second cross
hair which can be used to form a "box" around a region (see 2D spectrum
above). Once a suitable region has been chosen the Expand button can be clicked
to give the pertinent expansion.
The middle button can be used to change the vertical slice through the "topographic"
surface and can also be used to change the color map or contour levels.
To plot any region go to the PLot tab and click the Autoplot button which will cause
the 2D region to be plotted along with the 1D proton spectrum that was collected
before the COSY.
Practice reprocessing the data using different weighting functions and by turning
off the Linear Prdiction radio button. How does the application of a weighting function
such as the exponential line broadening in either dimensioin affect the spectrum.
Weighting functions can be observed in either dimension by transforming the dimensions
one at a time. Start by displaying the directly detected dimension by typing df(1)
wft(1) on the command line. Now press the Interactive button under the F2 processing
section to bring up the following window:
Change the weighting functions in a way analogous to that used in the 1D spectra.
When a new function has been applied click the Transform F2 Only button in the Processing
tab. This will transform t2 into a frequency domain and present a map of the interferograms
in t1. New weighting functions can be applied in the F1 transform by setting the
window funtion via the pull down menu. Click Full 2D transform to see the resulting