Note: Items shown in bold refer to the Varian vnmr parameters or commands.
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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. One can also use the s2pul sequence to perform relaxation and kinetic measurements.
In the standard 2 pulse sequence, you can define two different delays (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 300), or 1H while observing any other nucleus (on the Unity 500 and UnityPlus 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'. dm='nnn' would turn the decoupler off during all three periods. 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.
| decoupler mode | dm='nnn' to 'yyy' | dm can be 'n' or 'y' during equilibration, mixing and acquisition. The decoupler will be off if dm = 'n' and on if dm = 'y' during the period. |
| decoupler modulation method | dmm='ccc' to 'www' | dmm switches the decoupler irradiation from continuous wave ('c') to broad-band waltz modulation ('w') during each of the three periods. You can also use other decoupling modes, e.g. garp ('g'), or user programmed modes, during periods A, B or C. |
The combined use of dmm, dm, d1, p1 and d2 in a single pulse sequence enables a surprising number of useful nmr experiments to be easily performed, as shown in the following sections. T1, kinetics, and NOE measurements are covered in detail in separate manuals.
The most basic NMR acquisition requires a single pulse, followed by acquisition of the spectra. The spectra are acquired without decoupling. The complete sequence is repeated nt times, until the desired signal to noise is obtained.
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d1=0 d2=0 p1=0 pw=90 degrees or less dm='nnn' dmm='ccc' (not important, since the decoupler is off at all times) |
The transmitter Tx is set to the frequency of the nucleus of interest, e.g., 1H, 13C, 31P, etc.
Quantitative 1H spectra also require a single, 90° pulse and a delay d1 that is allows the magnetization to return to its original equilibrium state. The spectra are acquired without decoupling. The complete sequence is repeated nt times, until the desired signal to noise is obtained.
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d1=5 to 60 sec or more (>5 times T1) d2=0 p1=0 pw=90° (entered in usec. Type pw90? to see the 90 degree pulse time. Alternatively, you can enter the desired pulse angle by typing |
The simple quantitative sequence can be used to perform kinetics measurements as well. Simply make an array of experiments. For example, 8 spectra of sixteen transients could be acquired by setting nt=16,16,16,16,16,16,16,16. One could also make an array of spectra, but wait a fixed time between acquiring successive spectra using a pre-acquisition delay, pad. For example, 5 minutes could be left between successive spectra by typing nt=1, and pad=0,300,300,300, etc. (use pad=0 to acquire the fid with no delay).
C. Inversion Recovery T1 Measurements
T1 measurements require a long (5x T1) delay of time d1, followed by a 180° inversion pulse p1. The magnetization is allowed to recover for a time d2. The residual magnetization is then examines by a quantitative 90° pulse, pw. The sequence is repeated for nt transients at various values of d2.
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d1=5 x T1 p1=180° d2=array from ca. 0.01 x T1 to 3-5 x T1 pw=90° |
In the inversion-recovery T1 measurement, d2 is arrayed to cover the exponential relaxation of the magnetization. The command dot1 will generate an appropriate array of d2 delays, based upon guesses of the shortest and longest relaxations and the overall desired experiment time. dot1 will also set p1, d1 and pw to the proper values.
D. Homonuclear NOE Measurement
In the homonuclear NOE experiment a spectrum is acquired with single-frequency (selective) CW irradiation of one resonance. This spectrum is subtracted from a second spectra acquired without irradiation of the peak.
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d1=5 x T1 (typically 5-20 s) d2=1 to 5 x T1 pw=90° or less dm='nyn' dmm='ccc' |
The homonuclear NOE measurement requires an array of decoupler frequencies. The first spectrum is acquired with the decoupler off-resonance from any peaks. Successive spectra are acquired with irradiation of a single peak for a time d2. The decoupler is turned off and the transient is acquired after the pulse pw. The spin-system is allowed to return to equilibrium during the time d1. Setup, acquisition, and processing of the homonuclear NOE measurement is covered in detail in a separate handout.
E. The Four Basic Carbon (x-nucleus) Experiments
Carbon is spin one-half and strongly couples to directly attached protons (J = 80-160 Hz). Even protons removed by several bonds lengths can show couplings to carbon on the order of 1 to 10 Hz . Hence, most carbon spectra are obtained with 1H decoupling, in order to remove splitting from protons. 1H decoupling will usually lead to a significant increase in the signal intensities of the carbon signals as well, from NOE enhancement. The increased signal strength caused by NOE is usually desired, due to the low sensitivity of most carbon experiments.
A disadvantage of simple 1H decoupling is that it is impossible to make quantitive measurements from these spectra without first knowing the strength of the NOE values for each resonance. In addition, it is sometimes desirable to observe all the carbon couplings. Unfortunately, this experiment suffers from extremely low sensitivity: the peaks are split into multiplets and there is no NOE enhancement. We can take advantage of the ability to gate the decoupler on and off at various periods to assist with these different meausurements.
Four basic ways of acquiring 13C (or other x-nuclei as well) can be generated from a combination of decoupling and NOE effects. Spectra can be obtained with decoupling and NOE; no decoupling and full NOE; decoupling and no NOE, or no decoupling with no NOE. These four experiments are shown in the following figure. These four experiments take advantage of the ability to easily turn on or off the decoupler at different periods in the NMR experiment, using the dm command.
In example A., the broad-band decoupling is used throughout the complete experiment to give a fully decoupled carbon spectrum with full NOE (dm='yyy', dmm='www'). The delay d1 is optional and may be used in conjunction with the pulse width pw to optimize sensitivity.
The NOE enhancement is retained in example B, however, the spectra is acquired without proton decoupling, i.e., the carbons will all show couplings or splittings from the attached protons (dm='yyn', dmm='wwc'). The delay d1 is required in this example and should be 1-5 x T1 of the protons in order to build up the NOE enhancement. The pulse sequence in example C., (dm='nnn', dmm='ccc') would yield a fully coupled carbon spectrum with no NOE enhancement. If d1 was set to 5 X T1 for carbons, this experiment would give quantitative integrals for the resulting carbon multiplets.
Example C. is the least sensitive of the four experiments and can easily take 10 to 100 times longer to acquire than the decoupled, NOE enhanced spectrum (example A).
Example D. shows how to acquire a quantitative carbon experiment (pw=90°, dm='nny', dmm='ccw', d1=5 x T1). The delay d1 is set to 5 times the T1 of the 13C atoms of interest. The pulse width pw is set to 90° and the decoupler is turned on only during acquisition to prevent buildup of NOE enhancement.
Examples of these four 13C experiments, performed on the Gemini-300 using a solution of 30% Menthol in CDCl3 (nt=1, d1=10) are shown below.
