Note: Items shown in bold refer to the Varian vnmr parameters or commands.
The Varian software on both the Gemini and Unity systems uses a simple, but extremely flexible standard pulse sequence, called s2pul. Although literally hundreds of other pulse sequences are available, the simple, basic s2pul can be used to carry out routine NMR measurements, obtain quantitative nmr spectra, perform homonuclear and heteronuclear decoupling, NOE, and solvent suppression experiments. Also s2pul sequence to perform relaxation and kinetic measurements can be made.
A diagram of the s2pul sequence is shown below. In the standard 2 pulse sequence, two different delays can be defined (d1 and d2), and two different pulses (the observation pulse pw and a second pulse p1). The delays and pulses can be used to define three time periods. The d1 delay is used in period A to allow the spins to return to equilibrium and prepare the sample for subsequent pulses. The pulse p1 and the delay d2 occur during period B and can be used to create mixing of magnetization and allow for evolution of spin-coupling information. During period C, the signal is acquired after the observation pulse pw.
The pulses and delays in the s2pul sequence are carried out using a transmitter tuned to the observe frequency of the nucleus of interest. A second transmitter, called the decoupler, is available and can be used to perform simultaneous broad-band or selective excitation of the nucleus being observed. The decoupler can be used to irradiate 1H while observing 13C (on the Gemini 200), or 1H while observing any other nucleus (on the Unity 400). On the Varian systems, the decoupler can easily be turned on or off during any of the three periods A, B, and C. The decoupler can also be set to perform selective excitation during one period and then change to broad-band excitation during a different period.
The "status" of the decoupler is controlled by the parameters dm (decoupler mode) and dmm (decoupler modulation method). During each period A, B, and C, dm can be 'n' (decoupler off) or 'y' (decoupler on). For example, to turn the decoupler on during equilibration and acquisition, or periods A and C, dm should be set to dm='yny'. Then dm='nnn' would turn the decoupler off during all three periods while: dm='yyy' would leave the decoupler on at all times. Similarly, the decoupler modulation method can be set to continuous wave (cw or selective irradiation) in each period by setting dmm='ccc'. Waltz-modulation (broad-band) excitation is set using dm='www'. Any combination of cw ('c') and broad-band waltz ('w') decoupling is allowed during each status period. For example, one could selectively excite a single frequency during the equilibration and mixing periods and then switch to broad-band decoupling using dmm='ccw' and dm='yyy'. Also note that d1, p1 and d2 can be zero, enabling the user to eliminate periods A and B from the pulse sequence program.
Setting Up Parameters -- On entering NMR software the user has joined an experiment with its particular set of parameters. The parameters will be from the last user. To repeat with the same conditions there is no need to "setup" parameters. To change one or two parameters they can be typed in e.g NT=18. The parameters are displayed on screen with DG, DGS (special), or DG1 (display parameters). For arrayed parameters use DA1, DA2, and DA3. There are several ways to set up experiments 'from scratch:'
The S2PUL pulse sequence is set up and begin by entering values for P1, PW, D1, and an array of values for D2 or use the macro DOT1 which asks a series of question to help set up the experiment.
Type DT1 (LineNumbers) to display the calculated relaxation times T1 for a series of lines, T1(LineNumbers) prints the information. DELS(ArrayMembers) command removes "bad" data points after a T1 or T2 calculation has been performed. Spectra that have been removed can be added back with ADDS.2-D COSY (Correlated Spectroscopy) Experiments
This 2D experiment is composed of a 90o pulse that creates magnetization in the transverse plane. During the evolution time, the variable delay t1 is incremented systematically in order to sample the spectral width indirectly. Following this variable time period, a second pulse mixes the spin states, transferring magnetization between coupled spins. The spectra is then acquired during t2 (detection time). After a double Fourier Transformation, a spectra like the one below is obtained showing a diagonal component (for magnetization that did not exchange magnetization) and cross peaks (off-diagonal) for nuclei exchanging magnetization through scalar coupling. The data is usually symmetrical respect to the diagonal and therefore the data can be symmetrized as part of the processing to improve the quality (care must be taken here to make sure that by getting rid of the non-symmetrical artifacts we are not also getting rid of precious information that might not be totally symmetrical). The data is usually acquired in a phase insensitive (magnitude mode) manner, avoiding the difficulty to phase a 2D data set. This phase insensitive mode gives rise to very broad line shape that can be sharpen using sine-bell or pseudo-echo shaping processing method.
Variations of the COSY experiment
Obtaining a 1H COSY (Homonuclear Correlated Spectroscopy) Spectrum Unity 400
The VNMR software interface uses both entry commands and menu driven commands. All entry commands will appear in boldface, while the menu driven commands will appear as 'select (button)'. The function keys (F1...F12) may be used instead of clicking on the menu buttons. Some buttons do not have the same appearance those in the menu bar, such as the lock on/off commands and toggling between shim parameters; they may be activated by clicking on the highlighted text.
Most functions can be executed with either typed commands or menu commands. This procedure is intended for an entry-level user.
1. Acquire a Proton Spectrum -- The first step in setting up for a COSY spectrum is to acquire a proton spectrum of the sample and optimize the spectral width. Take a 1H NMR spectrum of the sample in the normal way. After data acquisition is finished, display the full spectrum (all 15 PPM) and put up two cursors. Bracket the left-most and right-most peaks in the spectrum with the cursors, leaving about 0.5 PPM of noise between each cursor and the nearest peak.
2. Next, type movesw. The movesw command will adjust the spectral width SW and the transmitter frequency to correspond to the region defined by the two cursors. WARNING: DO NOT TYPE movesw MORE THAN ONCE IN THE SAME EXPERIMENT WITHOUT RE-ACQUIRING THE SPECTRUM.
3. Defined the optimum spectral window for the COSY experiment by taking a simple proton spectrum and narrowing the sweep width, with movesw, move these new parameters from the current experiment (exp1) to a new experiment, which will be used to acquire and process the COSY spectrum. Move the acquisition parameters from the proton experiment (exp1) to experiment 2 (exp2) by typing mp(1,2). This will copy the current acquisition parameters into exp2. Next, join exp2 by typing jexp2.
4. Once the 1D proton parameters have been copied into exp2, they can be transformed into the acquisition parameter set suitable for acquiring a COSY spectrum simply by typing cosy. The cosy command will load the pulse sequence required to obtain a COSY spectrum, and will convert the optimized 1D parameters into the 2D parameter set.
Select the sweep width -- Because the COSY experiment is 2D, the resolution depends on the square of the sweep width--to take 64 increments over a 3 ppm range requires 128 increments over a 6 ppm range. (double the time). Taking 64 increments over a 6 ppm range results in half the resolution. After determining the 90 degree pulse width, return to the normal 1H experiment and acquire a spectrum and choose the pulse width: Type vp=0 full nt=8 ga
To the displayed spectrum, select an area for the 2D experiment by bracketing this region with the two red cursors (left button, left cursor; right button, right cursor). Do not expand the spectrum. Instead, type movesw to change the values of sw and tof to match the selected region. These values may be calculated, but the movesw command is much faster.
5. Setup COSY Experiment -- Type cosy. This transfers all parameters (sw, tof, pw) to the cosy experiment. Type dg. This displays the experimental parameters. Note the values of np, nt, ni. Type time. This will calculate the length of the experiment. Adjust np, nt, and ni to make the experiment run a proper length of time (i.e. there is only 20 minutes left, or an 8 hour overnight experiment). This is important because, unlike 13C experiments, data acquired at the end of the run is as important as data at the beginning. Collect a full set of data.
Note: np is simply a parameter regarding the digital resolution. Because the data size is the square of the np value, choose a number less than 2000. (Typically 256, 512, 1024, or 2048). This has a small effect on the length of the experiment.
More importantly, nt and ni need to be optimized. The value of nt reflects the inherent signal-to-noise of the fids, just like 1D experiments. More is better. ni reflects the number of increments that the delay time tau will be divided into. This is important in getting good resolution in cross peaks. More is definitely better here. Because the time is proportional to nt x ni, vastly increasing one will sacrifice the other. Typical values include:
nt=8, ni=64; nt=4, ni=128, nt=16, ni=32 (note: these values give the same time but different results)
Always check the time after fiddling with nt and ni. This is the final arbiter of values. Make sure there is enough time to complete the experiment.
If the experimental range is small and the compound is simple enough, A 5 minute COSY run could be successful. Typically, only 10-20 minute runs are best. Rarely will there be a need to perform a COSY for more than two hours.
Acquiring the FID
go will start the experiment.
The experiment will run until it completes the number of scans indicated in the parameters. After acquisition, the computer will return 'Acquisition Complete' and the status indicator in upper left corner of the acquisition status window will read idle.
Processing the FID
wft2d weight and Fourier transform (2D)
The spectrum will be displayed automatically.
Displaying the Spectrum
Cursors allow zooming. Use both left and right cursors to define a box. Select the button to expand this area. Select the button to display the full spectrum.
Single cursor can be used to select individual peaks. The location of the peak is displayed at the bottom of the screen.
Use the double cursors to line up diagonal peaks with their cross peaks. Keeping a 1D spectrum handy to annotate the changes on the 2D surface may helps significantly.
Click in the spectrum to change scale. This can be tricky to control; just note the value of vs parameter and enter a new value, then press the "redraw" button.
The color panel at the right controls the extent of visible signals. Click on the colors to turn them on and off. This can be used to display only the strongest peaks, removing the noise. Work with both high and low noise; mainly because small cross peaks may prove important for interpretation. Usually, though they are distracting enough to be discarded. Always keep in mind this possibility and simply be comfortable manipulating the intensity values.
Other tricks including peak picking are available. Please refer to the Varian manuals.
Setting a Reference Peak
Place the single cursor on a peak to be referenced.
rl(xxxp) will set the position of the cursor to xxx ppm. rl1(xxxp) will set the position of the second axis to xxx ppm.
Printing the Spectrum
Usually there is no need to print spectra. Annotate 1D spectra and save the COSY file for future reference. Plot the spectrum, using the "autoplot" button.
Detailed instructions for plots, including projected 1D spectra, peak picking, contour plots, etc. are available.
Saving the FID
svf('filename') will save the file as filename. rt('filename') will retrieve filename. ls will list all files in the user's directory under Unix.