Pulses shaped/selective

Frequency-selective REDOR (fsREDOR) is a very powerful technique developed for the study of 13C and 15N uniformly labeled peptides or proteins [92]. The basic idea of this technique is to combine REDOR and soft n pulses to recouple a selected 13C-15N dipole-dipole interaction in a multiple-spin system. Usually one could use Gaussian shaped pulses to achieve the required selective n inversions. Other band selective shaped pulses have been developed for a more uniform excitation profile [93]. In its original implementation, fsREDOR was used to extract the intemuclear distances of several model crystalline compounds [92], In the past few years, this technique has proven to be very useful for the study of amyloid fibrils as well. For the Ure2p10 39 fibril samples containing 13C and 15N uniformly... 

In this chapter, the discussion will be focused on the ID TOCSY (TO-tal Correlation SpectroscopY) [2] experiment, which, together with ID NOESY, is probably the most frequently and routinely used selective ID experiment for elucidating the spin-spin coupling network, and obtaining homonuclear coupling constants. We will first review the development of this technique and the essential features of the pulse sequence. In the second section, we will discuss the practical aspects of this experiment, including the choice of the selective (shaped) pulse, the phase difference of the hard and soft pulses, and the use of the z-filter. The application of the ID TOCSY pulse sequence will be illustrated by examples in oligosaccharides, peptides and mixtures in Section 3. Finally, modifications and extensions of the basic ID TOCSY experiment and their applications will be reviewed briefly in Section 4. 

To understand selective (shaped) pulses and the spin lock, we need to look in detail at the effect of pulses on spins as a function of their resonant frequency, v0, that is to say the position of a resonance within the spectral window. 

Figure 9.14. Simulated excitation profiles of selected shaped pulses (of 10 ms duration) see Table 9.3. The inversion profiles (lower trace) were simulated with a 180(soft)-90(hard) sequence.

Gaussian pulses are frequently applied as soft pulses in modern ID, 2D, and 3D NMR experiments. The power in such pulses is adjusted in milliwatts. Hard" pulses, on the other hand, are short-duration pulses (duration in microseconds), with their power adjusted in the 1-100 W range. Figures 1.15 and 1.16 illustrate schematically the excitation profiles of hard and soft pulses, respectively. Readers wishing to know more about the use of shaped pulses for frequency-selective excitation in modern NMR experiments are referred to an excellent review on the subject (Kessler et ai, 1991). 

Fig. 14.4 Pulse sequences used for the experiments described in this chapter. A [ N HJ-HSQC with water flip back and PFGs. The shaped pulse on the proton channel is a sine-shaped, 1.5 ms soft pulse all other pulses are hard pulses. Gradients are applied as square or sine-shaped pulses. The sign of the last gradient is reversed for anti-echo selection together with the sign of phase 6. B CPMG sequence. C bpPFGLED sequence. The delay T denotes the diffusion delay. Typically, r is set to 1 ms, T to 50-100 ms and Te to 1.2 ms.

D NOE-pumping experiment. E Reverse NOE pumping experiment (bottom) and reference experiment (top). F e-PHOGSY NOESY sequence. The water-selective 180° pulse sandwiched by the first two gradients has a gaussian shape and a duration of 40-50 ms. The mixing time is approx. 2 s. For further details, see Refs. [29, 30]. 

Selective saturation (using shaped pulses) of all the aromatic ring CH resonances together since they show up generally in a short window of 6.5-8.5 ppm. The Q-matrix is easily set up for this case. 

Similarly to non-selective experiments, the first operation needed to perform experiments involving selective pulses is the transformation of longitudinal order (Zeeman polarization 1 ) into transverse magnetization or ly). This can be achieved by a selective excitation pulse. The first successful shaped pulse described in the literature is the Gaussian 90° pulse [1]. This analytical function has been chosen because its Fourier transform is also a Gaussian. In a first order approximation, the Fourier transform of a time-domain envelope can be considered to describe the frequency response of the shaped pulse. This amounts to say that the response of the spin system to a radio-frequency (rf) pulse is linear. An exact description of the... 

The peak rf amplitude required to achieve optimum excitation with a selective excitation pulse is given in comparison to the rf amplitude required to achieve an on-resonance 90° flip-angle with a selective rectangular pulse, the simplest conceivable shape. 

Fig. 1. Computer simulations of four selective excitation pulses. (Top) Pulse shapes. From left to right 90° rectangular pulse, 270° Gaussian truncated at 2.5%, Quaternion cascade Q, and E-BURP-1. The vertical axis shows the relative rf amplitudes, whereas the horizontal axis shows the time. (Middle) Trajectories of Cartesian operators in the rotating frame...

For selective irradiations with a flip angle of 180°, one can distinguish two groups of shaped pulses inversion pulses, which change the sign of Zeeman... 

The principle of multiple selective excitation has been incorporated into a few ID and 2D experiments, the schemes of which are shown below (fig. 1). Depending on the experiment, either a DANTE pulse train (ID TOCSY [2]), frequency selective 180° pulses (ID NOE [3], ID INADEQUATE [4], ID C/H COSY [5] and 2D TOCSY-COSY [6]) or frequency selective 90° pulses (2D HMBC [11]) are applied to selectively perturb and uniquely label selected spins. Besides the DANTE pulse , composed itself of a series of non-selective rectangular pulses, Gaussian-shaped 180° and... 

The double-selective TOCSY-ROESY and TOCSY-NOESY techniques are particularly useful. They allow one to measure NOE and ROE correlations in spectra with high degree of overlap as often found in carbohydrates. In addition to the DANTE, DANTE-Z [66], and Gaussian pulses as described earlier for selective excitation, self-refocusing shaped pulses such as BURP (EBURP and UBURP) [67] have also been used for this purpose [64]. 

X/Y coherence transfer steps as in the original HNCA experiment.41 As in the previous case, X nuclei are selectively irradiated by low power rectangular or shaped pulses, and coherence selection is accomplished by the matched pulsed field gradients Gb G2 and further assisted by a spoil gradient Gs. Owing to the need to avoid -pulses in the X-channel, the spectra are processed in magnitude mode in the fft dimension. 

Fig. 3. Experimental demonstration of two-photon selective microscopy. The HPTS-labeled sample being imaged has acidic (bottom side) and a basic (top side) regions. Images were obtained with (A) 23-fs transform-limited pulses centered at 842 nm, and (B and C) phase shaped pulses optimized for selective excitation.

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