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homo

Decoupler nucleus: H1, C13, N15, and so on. This determines the basic frequency of the decoupler irradiation (300.0, 75.4, 30.4 MHz, etc.) Decoupler offset: This sets the exact frequency (chemical shift) of the decoupler irradiation in hertz.

Decoupler mode: This determines when the decoupler is on or off during the pulse sequence (e.g., "nny" for on, on, off).

Decoupler power: Power level of the decoupler irradiation in decibels (dB) (increasing power from 0 to 63 for Varian, from 120 to —6 for Bruker) Decoupler modulation: This defines the decoupling sequence for composite pulse decoupling (e.g., Varian 'w' for waltz-16)

Decoupler modulation frequency. This sets the 90° pulse width at the power level used for decoupling (90° pulse = dcpd2 = 1/dmf) Homonuclear: Set to 'y' for homonuclear (1H-1H) decoupling, or 'n' for heteronuclear (e.g., 1H-13C) decoupling.

Bruker has no corresponding parameter for dm and homo because these options are written into each pulse program.

The Varian parameter dmm determines whether the decoupler output is a simple continuous irradiation (dmm ="c") or a pulsed waltz-16 modulation (dmm ="w"). For nonselective ("broadband") decoupling such as that desired for a 1D 13C spectrum, the waltz-16 mode is used to minimize power requirements and maximize the range ("bandwidth") of chemical shifts decoupled. For selective decoupling, the continuous mode is used to minimize the range of chemical shifts affected. For Bruker the choice between continuous wave (cw) and composite pulse decoupling (cpd) is coded into the pulse program; the parameter cpdprg2 defines the sequence used for composite pulse decoupling (e.g., waltz-16). The Varian dmf (decoupler modulation frequency) parameter is used to set the duration of pulses (e.g., 90° pulse, 270° pulse) in the waltz-16 sequence. It is determined by calibration of the 90° pulse width at the power level dpwr and is defined as the reciprocal of the 90° pulse width: dmf = 1/tp(90) in units of hertz. From the example above (100 ^s 90° pulse for waltz-16), we would use dmf = 1/(100 ^s) = 10,000 Hz. The 90° pulse is much longer than a hard pulse (~10 ^s) because we use much lower power for decoupling. Note that the decoupler field strength in hertz is one fourth of dmf (typical yB2/2n = dmf/4 = 2500 Hz). Bruker uses the parameter dcpd2 for the 90° degree pulse width calibrated at power level pl17 (decoupler power level). The decoupler frequency is set by the Varian parameter dof (decoupler offset) and Bruker parameter o2 (oh-two, offset channel 2), which function just like tof (transmitter offset) and o1 (oh-one, offset channel 1), respectively.

The decoupler power (Varian dpwr, Bruker pl17) is set according to the desired effect of the decoupler irradiation. Bruker uses a decibel scale for RF power that decreases as power increases (120 to -6 dB), and Varian uses a decibel scale that increases as power increases (0 to 63 dB). For homonuclear (i.e., XH-XH) NOE experiments, a very low power (5 dB Varian, 58 dB Bruker) is used to maximize selectivity—only a "simmer" is required to equalize populations. For selective decoupling, values of 10-15 (Varian) or 48-53 (Bruker) are typical—small enough to be selective but powerful enough to maintain the "rolling boil" necessary for decoupling. For broadband (nonselective) decoupling (e.g., waltz-16), a power level of 40 (Varian) or 23 (Bruker) is typical, adjusted to obtain good decoupling over the entire range (e.g., 5 ppm ± 6 ppm: -1 to 11 ppm) of proton chemical shifts. For each setting of decoupler power, the 90° pulse must be measured and dmf (1/t90) or dcpd2 (t90) set appropriately.

There are more advanced experiments such as DEPT (Chapter 7) that observe 13 C and use the decoupler to supply high power, short duration ("hard") pulses at the 1H frequency. This requires full power from the decoupler, but the parameters dpwr and pl17 are avoided for these pulses. Setting decoupler power to the maximum might lead to disastrous mistakes because the decoupler can only deliver full power for short (~ 10 ^s) periods of time without burning up the decoupler, the probe, and the sample. Instead, the parameters pp (Varian) and p3 (Bruker) are used for the 90° pulse width for decoupler hard pulses and pplvl (Varian) and pl2 (Bruker) indicate the power level for short-duration high-power decoupler pulses.

4.7 THE NUCLEAR OVERHAUSER EFFECT (NOE)

The population distribution of a nucleus (difference between populations in the upper spin state and the lower spin state) can be affected by the population distributions of other nuclei that are nearby in space. Experimentally, one can observe an enhancement of the population difference of one nucleus by saturating (equalizing the populations of) a nearby nucleus (Fig. 4.12). In the figure, filled circles represent a slight excess of population (+5) and open circles represent a slight deficit (-5). Irradiation of one proton signal (Ha) equalizes its populations across the a ^ j transition (Fig. 4.12, center), and over a period of time this perturbation "propagates" through space to a nearby proton (Hb), which

Figure 4.12

experiences a population perturbation in the opposite sense: an increase in population difference (Fig. 4.12, right). An enhanced population difference means a larger net magnetization along the z axis (Mz > Mo) and can be observed by applying a 90° pulse to the affected nucleus and observing its NMR signal, which will also be enhanced. The intensity of this effect dies off very quickly with increasing distance between the saturated nucleus and the observed nucleus: the exact dependence is 1/r6, where r is the distance between the nuclei. This is extremely important for proton-proton interactions because it allows distances between individual atoms in a molecule to be measured. This strategy has led to the accurate determination of 3D structures of proteins and nucleic acids in aqueous solution, so that NMR now rivals X-ray crystallography as a method for defining the precise conformations of biomolecules.

Heteronuclear NOEs can also be observed; for example, between 1H and 13 C nuclei (Fig. 4.13). Continuous irradiation of the proton signal during the relaxation delay leads over time to an increase in the population difference (and net z-magnetization) of the13 C bonded to that proton. This builds up and finally levels off at a steady state, where relaxation exactly balances the enhancement from the proton. At this point the 13C pulse rotates this enhanced z-magnetization into the x-y plane where it precesses and induces an enhanced signal in the probe coil. After Fourier transformation we have an enhanced peak height in the 13 C spectrum (Fig. 4.13). We want all of the 13C signals to get this benefit, so waltz-16 decoupling is used to irradiate the protons, covering the entire range of XH chemical shifts. Because saturation of protons is typically carried out during acquisition of 13C signals anyway to eliminate the effects of XH-13C J coupling, it is convenient to continue this saturation throughout the whole experiment, including during the relaxation delay. The effect is that a heteronuclear NOE builds up on the 13 C nuclei during the relaxation delay, enhancing their z magnetization and giving a stronger signal in the FID after this z magnetization is rotated into the x-y plane by the observe pulse. This gives the 13 C signals a much-needed increase in the signal-to-noise ratio. It is not used to measure distances because the majority of the enhancement comes from directly bound protons, and this covalent bond distance is already known.

Figure 4.13

4.7.1 Comparison of NOE and Decoupling

It is important to recognize that the power level required for saturation (equalization of populations in the two energy levels) of nuclei, which causes the NOE, is much less than that required for decoupling. Decoupling requires not just equalization of populations but a situation where each 1H nucleus jumps back and forth rapidly between the two levels. It is sort of like simmering the spins versus a raging boil. Continuous saturation causes the NOE to "build up" to a steady-state level over a period of time on the order of T1. The NOE manifests itself as an enhancement of Mz in the target nucleus, and the effect dies off after saturation is discontinued with a time constant on the order of T1. The irradiation in an NOE experiment occurs during the relaxation delay and before the exciting 90o pulse. Decoupling is effective only during the acquisition period because it averages out the effect of the spin state of other nuclei on the precession frequency of the observed nucleus. Thus to decouple the irradiation must occur during acquisition of the FID. Decoupling manifests itself as a reduction or elimination of the J coupling, and the effect stops immediately after the decoupler is turned off.

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