Solvent pulse sequence

One problem in recording nOe spectra of proteins in aqueous solutions is the presence of a water signal. Soft NOESY produces minimal excitation of the solvent signal. The pulse sequence used is shown in Fig. 7.18 (Oschki-nat et al, 1988 Oschkinat and Bermel, 1989). 

Pre-saturation In this technique prior to data acquisition, a highly selective low-power rf pulse irradiates the solvent signals for 0.5 to 2 s to saturate them. No irradiation should occur during the data acquisition. This method relies on the phenomenon that nuclei which have equal populations in the ground and excited states are unable to relax and do not contribute to the FID after pulse irradiation. This is an effective pulse sequence of NOESY-type pre-saturation that consists of three 900 pulses RD - 900 - tx - 900 - tm - 90° - FID, where RD is the relaxation delay and t and tm are the presaturation times. 

Other even more cunning methods have been devised to suppress the water signal in samples that have a large water content (e.g., bio-fluid samples) such as the WET and the WATERGATE pulse sequences. Other sequences have been devised to cope with signals from carbon-bound hydrogens. Some of these actually collapse the 13C satellites into the main 12C peak prior to suppression. Such a sequence would be useful if you were forced to acquire a spectrum in a nondeuterated solvent. 

Figure 8.3 Illustration of HR-MAS techniques applied to a resin-bound trisaccharide (a) static XH spectrum of the solvent swollen sample (b) XH spectrum with magic-angle spinning at 3.5 kHz (c) H spectrum with MAS and spin echo pulse sequence.

Fig. 21.1 Pulse sequence to selectively observe solvent-exposed amide protons with TROSY (SEA-TROSY). Narrow and thin bars represent 90 and 180° rf pulses, respectively. Unless specified otherwise, pulse phases are along the x axis. The pulsed field gradients are of 500 ms duration with strengths of gi =20 G cm-1, g2 = 30 G errf1, g3 = 40 G errf1, g"4 = 15 G cm-1, g5 = 55 G errf1. The bipolar gradient gj is 0.5 G errf1 and is used to avoid...

The D-HMBC pulse sequence can also be used in combination with the pulse field gradient (PFG) technique [12]. Figure 5(c) shows the successful observation of cross peaks between the methyl group at C-5 of an oxazole unit and adjacent carbons in promothiocin. These cross peaks are hidden by the strong t noise of the solvent peak in the HMBC and D-HMBC spectra. The above results clearly indicate that D-HMBC is a quite useful technique for structural studies of complicated natural products. 

Fig. 13. (a) 1H/(31P)/15N correlation of a mixture of Mes P( = NH) = NMes (compd. 2, Mes = 2,4,6-tri-t-butylphenyl) and Mes P(NHMes )-N1 = N2 = N3 (compd. 3) with correlations involving the iVH and aromatic protons in the P-Mes substituents. The spectrum was obtained with the pulse sequence shown in Fig. 11a. The tx noise around S1H = 5.1 is due to a solvent signal (CH2C12) which is 4 105 times more intense than that of the 15N-satellites of the iVH-resonance of 3. (b) Expansion of a -detected 2D-/P N-resolved spectrum of the same mixture with correlations of the aromatic protons in the P-Mes -substituents as obtained with the pulse sequence shown in Fig. 12. 2q cross-sections of the 2D-spectrum at the chemical shifts of the aromatic protons of 2 and 3 are given in (c) and (d), respectively, and reveal the presence of one (2) and three (3) resolved JP N couplings. Reproduced from Ref. 43 by permission of John Wiley Sons.

Fig. 7. 300 MHz H HRMAS NMR spectra of a resin suspension swollen with DMF-dq from a reaction mixture and spun at 4 kHz. The spectrum (A) was obtained with a single-pulse sequence. The spectra in (B), liberated from the reaction vessel at the times indicated, were obtained using a diffusion filter to reduce the signals from non-bound species. Note the excellent suppression of the solvent DMF peak at 8 ppm. Reproduced with permission from Ref. 75. Copyright 2000 American Chemical Society.

The principle of presaturation relies on the phenomenon that nuclei which are unable to relax, because their population in the ground state a and the excited state (3 is the same, do not contribute to the free induction decay after pulse irradiation. Prior to data acquisition, a highly selective low-power pulse irradiates the desired solvent signals for 0.5 to 2 s, thus leading to saturation of the solvent signal frequency. During data acquisition, no irradiation should occur. NOESY-type presaturation is an effective pulse sequence of presaturation. The pulse sequence consits of three 90° pulses (similar to the first increment of a NOESY experiment) ... 

Figure 1.16 Representation of the WET pulse sequence for multiple solvent suppression...

Direct on-line coupling of an NMR spectrometer as a detector for chromatographic separation, analogous to the use of MS for such applications, has required the development of technical features such as flow-probe hardware, efficient NMR solvent suppression pulse sequences and new software. 

This main difficulty in coupling HPLC to NMR spectroscopy is faced by methods known as solvent suppression techniques, where the large solvent signals are reduced by special pulse sequences, switched prior to the information-selecting and acquisition pulses. Therefore, many efforts have been made to develop effective and minor-disturbing pulse sequences, such as presaturation, zero excitation and PFG-pulse sequences (WET) (see Chapter 1 and the following chapters). Despite the possibility of also suppressing several of the... 

DEPT, H-H COSY, TOCSY, short range C-H chemical shift correlation and long range C-H chemical shift correlation experiments were performed on Bruker AMX 360 and AM400 instruments using acetone-d6 as solvent. C-H chemical shift correlation experiments were carried out with inverse detection. Standard pulse sequence programs provided with the instruments were used for all 2-D experiments. 

H and 19F NMR spectra are recorded with a normal one-pulse sequence or, alternatively, the XH spectra are recorded with a sequence that allows simultaneous solvent suppression with presaturation (31) or a sequence that includes some other method of suppression 13C 1H and 1P 1H spectra are recorded with proton broadband (composite pulse) decoupling (32), and 31P spectra with gated proton decoupling (33). 

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