Spin purge pulses

The last 90° pulse on 13C acts as a purge pulse for the undesired dispersive magnetization.47,48 The function of the pulse is to convert any magnetization remaining antiphase with respect to the 13C spin into unobservable multiple-quantum coherence. This will provide cross peaks with pure lineshapes and with higher resolution, and consequently establishes reliable determination of coupling constants.47,48... 

Use of High Power Spin-Lock Purge Pulses in High Resolution NMR Spectroscopy... 

In the following, three different experiments are discussed, where short, high-power spin-lock pulses are used to purge the spectrum from undesired resonances. The experiments are (i) the HSQC experiment [5], (ii) experiments with C half-filter elements [6], and (iii) NOESY and ROESY experiments for the observation of water-protein NOEs [7]. In the first two experiments, spin-lock purge pulses are used to suppress the signals from... 

HSQC with spin-lock purge pulses... 

A single spin-lock purge pulse inserted into the HSQC pulse sequence defocuses most of the undesired proton magnetization while maintaining the proton magnetization of the protons bound to C or [8]. 

The magnetization from C-bound protons is suppressed by the spin-lock purge pulse. Denoting the operators of a proton 2-spin system as and Hb, the product operator calculation yields... 

In practice, the suppression of the signals from C-bound protons is not complete. In part, this arises from imperfections of the 180°(if) pulse in the delay r. If the chemical shift evolution is not refocused, pure proton terms are generated which pass the spin-lock purge pulse. Therefore, the suppression of the signals from C-bound protons is improved by applying the Excorcycle [11] phase cycle to this 180°(if) pulse [10]. To keep the phase cycle short, only the first two steps of Excorcycle can be used. The selection of the correlations is further improved by phase cycling... 

Since water protons are not bound to or nuclei, the water signal is also suppressed by the spin-lock purge pulse. In practice, the suppression of the water signal is sufficient to record HSQC spectra of protein samples dissolved in mixtures of 95% H20/5% D2O without any further water suppression scheme [12]. For optimum water suppression the carrier frequency must be at the frequency of the water resonance. On resonance, the phase of the water magnetization is not affected by imperfections of the first 180°(ff) pulse, so that no solvent magnetization ends up along the axis of the spin-lock purge pulse. 

Because of the favorable cross-peak multiplet fine-structure, the HSQC experiment offers superior spectral resolution over the HMQC (heteronuclear multiple quantum coherence) experiment [13, 14], On the other hand, the HMQC experiment works with fewer pulses and is thus less prone to pulse imperfections. The real advantage of the HSQC experiment is for measurements of samples at natural isotopic abundance and without the use of pulsed field gradients, since the HSQC experiment lends itself to purging with a spin-lock pulse. Spin-lock purging in the HMQC experiment... 

High Power Spin-Lock Purge Pulses... 

Selecting the C-bound protons before performing a homonuclear two-dimensional experiment enables to measure small heteronuclear coupling constants [16]. Such an experiment with a sample of natural isotopic abundance was first published by Otting and Wuthrich in 1990, where the half-filter element with spin-lock purge pulse was used to select the C-bound protons in a small protein in aqueous solution [6]. Later applications illustrated the usefulness of the same half-filter element with smaller molecules [17, 18]. 

Fig. 5. Pulse sequences of NOESY and ROESY with spin-lock purge pulses for water suppression. (A) NOESY pulse sequence. The spin-lock pulses are typically of length 0.5 ms and 2 ms, and r = 1/SW, where SW is the spectral width in the acquisition dimension. Phase cycle (pi = x,—x) 4>2 = 4 x,x,—x,—x) ...

Excellent results are routinely obtained when applying PFGs to the transverse C coherence in a C HMQC experiment which is conducted at natural isotopic abundance [27]. Under those circumstances, however, half of the coherence transfer pathways are rejected by the PFGs. More complicated acquisition schemes and additional pulses in the sequence are required to restore the full sensitivity [26, 28, 29]. Spin-lock purge pulses may not purge as well as PFGs, but they are easier to use and don t interfere with a phase-sensitive recording. 

This suppression scheme has been shown to work well together with HMQC experiments of small molecules at natural abundance. Even cleaner spectra are obtained, if the BIRD sequence is combined with HSQC experiments already containing a spin-lock purge pulse. Drawbacks of the BIRD pulse scheme are the fact that the relaxation delay between scans cannot be chosen freely anymore and that complete suppression of all C-bound proton signals is impossible, if they have different relaxation times. Furthermore, the BIRD pulse scheme is not applicable to molecules in the slow motional regime, since negative NOEs between the inverted proton spins and the non-inverted C-bound proton spins would reduce the magnetization of the latter. 

A number of methods have been developed to suppress contributions to the spectrum from zero-quantum coherence. Most of these utilise the property that zero-quantum coherence evolves in time, whereas z-magnetization does not. Thus, if several experiments in which the zero-quantum has been allowed to evolve for different times are co-added, cancellation of zero-quantum contributions to the spectrum will occur. Like phase cycling, such a method is time consuming and relies on a difference procedure. However, it has been shown that if a field gradient is combined with a period of spin-locking the coherences which give rise to these zero-quantum coherences can be dephased. Such a process is conveniently considered as a modified purging pulse. 

A purging pulse consists of a relatively long period of spin-locking, taken here to be applied along the x-axis. Magnetization not aligned along x will precess... 

The combination of spin-locking with a gradient pulse allows the implementation of essentially perfect purging pulses. Such a pulse could be used in a two-dimensional TOCSY experiment whose pulse sequence is shown below as (a). 

Pulse sequences using purging pulses which comprise a period of spin locking with a magnetic field gradient. The field gradient must be switched on and off in an adiabatic manner. 

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