Solvents signaling

Figure 5.37 (a) Conventional phase-sensitive COSY spectrum of basic pancreatic trypsin inhibitor, (b) Double-quantum filtered (DQF) phase-sensitive COSY spectrum of the same trypsin inhibitor, in which singlet resonances and solvent signal are largely suppressed. Notice how clean the spectrum is, especially in the region near the diagonal line. (Reprinted from Biochem. Biophys. Res. Comm. 117, M. Ranee, et al., 479, copyright (1983) with permission from Academic Press, Inc.)... 

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). 

Presaturation Selective irradiation of a nucleus prior to application of a nonselective pulse causes it to be saturated, so its resonance is suppressed. This technique may be employed for suppressing solvent signals. 

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. 

However, all these suppression techniques have a common disadvantage in that the desirable signals of interest lying under the solvent signal are also suppressed. 

Figure 7.22 shows the H NMR chromatogram (contour plot) of the separation of a 10% phthalate mixture in CH2CI2. The spectrum is almost free from interferences the NMR resolution is excellent, and it is possible to identify all plasticisers even at concentrations as low as 2%, which corresponds to 60 xg per component. In contrast, in on-line HPLC- H NMR separation the regions between 3.9-3.3 and 1.9-1.7 ppm are completely obscured by solvent signals. 

On-line SFC- H NMR has also been applied for the investigation of monomeric acrylates [456] SFC-NMR and HPFC-NMR were compared for both these acrylates [456] and phthalates [459]. Direct SFC-NMR coupling offers the advantage that the recorded continuous-flow H NMR spectrum is not obscured by solvent signals. 

Note that, apart from the solvent signal, two aromatic carbon signals (at 125 and 147 ppm) show no correlation because they are quaternary (i.e. not bonded to protons). 

HPLC requires much larger amounts of solvents, so that deuterated materials are too expensive instead we work with undeuterated HPLC quality solvents, the proton signals from which are suppressed using the so-called WET sequence, which also suppress the carbon-13 satellites of the solvent signals. 

Signal for two protons lies under solvent signal... 

Of course, you don t have to use either of the above standards at all. In the case of samples run in deutero chloroform/methanol and dimethyl sulfoxide, it is perfectly acceptable, and arguably preferable, to reference your spectra to the residual solvent signal (e.g., CD2HOH) which is unavoidable and always present in your spectrum (see Table 2.2). These signals are perfectly solid in terms of their shifts (in pure solvent systems) though the same cannot be said for the residual HOD signal in D2O and for this reason, we would advise adhering to TSP for all samples run in D20. 

A solvent-suppression method is normally required in NMR applications because the solvent signals, if not suppressed, may saturate the receiver and hinder... 

FIGURE 7.6 Extracted NMR spectra (600 MHz, 1.5 xL solenoidal microprobe) of the tocopherol homologues at the corresponding peak maxima residual solvent signals. (Reproduced with permission from Ref. 30. Copyright (2004) American Chemical Society.)... 

The increased solvent requirements for LC-NMR over tube NMR make the technique expensive if fully deuterated solvents are used. D2O is usually substituted for H2O in the eluent, mainly because of its low cost ( 150 per 1), but also because it provides a lock solvent for the NMR. Fully deuterated organic modifiers such as 3-acetonitrile may not be absolutely required, but in practice they are often also used in many pharmaceutical laboratories as they give reduced solvent signals. This reduces the need for solvent suppression, which lessens the risk that diagnostic resonances are also suppressed in error. 

In 1,1,2,2-tetrachlorethane solution, the spectra of thianthrene and trans-thianthrene 5,10-dioxide are temperature independent for the cis-isomer, the lower-field resonance shifts further downfield with increasing temperature while the signals for 2-, 3-, 7-, and 8- protons remain constant over a 200° range. In chloroform, small, apparant temperature-dependent shifts for thianthrene, at all positions, were attributed to temperature-dependent shifts of the reference solvent signal. Using this, it was shown... 

High Power Spin-Lock Purge Pulses 11. Solvent signal suppression by spin-lock pulses... 

The most frequently used NMR solvents for flavonoid analyses are hexadeuterodimethylsulf-oxide (DMSO-J6) and tetradeuteromethanol (CD3OD). Anthocyanins require the addition of an acid to ensure conversion to the flavylium form. For the analysis of relatively nonpolar flavonoids, solvents such as hexadeuteroacetone (acetone-J6), deuterochloroform (CDCI3), carbontetrachloride (CCI4), and pentadeuteropyridine have found some application. The choice of NMR solvent may depend on the solubility of the analyte, the temperature of the NMR experiments, solvent viscosity, and how easily the flavonoid can be recovered from the solvent after analysis. In recent years, the problem of overlap of solvent signals with key portions of the NMR spectrum has been reduced by solvent suppression and the application of improved 2D and 3D NMR techniques. 

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