Presaturation method

There are of course methods for eliminating (or at least partially eliminating) water signals in fact there are many such methods, and we will demonstrate the use of the simplest of these (which is quite effective), the so-called presaturation method. Before carrying out this experiment we need to determine the exact chemical shift of the water signal which we wish to suppress using a standard proton experiment (the computer software can help us here). 

Kg. 6. Comparison of the (A) Water-PRESS, (B) WATERGATE and (C) presaturation methods on a lysozyme sample (10 mM in W 10 H20 H20, pH 3.7). All three spectra were acquired at 310 K at 300 MHz and under the same conditions as far as possible (e.g. receiver gain and number of scans). In acquiring spectrum (C), an inversion delay (Dnp) of 2.58 s was used with a series of 0.5 ms z-gradient pulses with an intensity of about 1 G cm. It can be clearly seen that the intensities of the protein CH protons near the water frequency ( 4.5 ppm) are greatly attenuated in (A) and nearly absent in (B) compared to those in (C). (From Price et... 

Different principles are used (1) presaturation method presaturation of the signal, (2) jump-and-return method excitation of all signals followed by transfer of solvent coherence into not-detectable equilibrium magnetization of the solvent, (3) WATERGATE gradient driven destruction of solvent coherence... 

As discussed in the introduction to this section, the presaturation method is the simplest experiment since the suppression is based on either cw irradiation or a shaped selective pulse. The method is not recommended for solvents such as water or methanol if protons of the compound under investigation exchange with the presaturated protons of the solvent, since the resonance signal of these protons would also be presaturated and disappear. Consequently the presaturation method should not be used for sugars, proteins etc. if the amid, imid or hydroxyl protons are of particular interest. 

The jump-and-return method can also be implemented in 2D experiments in a similar manner to the presaturation method. Check it 5.23.4 shows the jump-and-return method as part of a 2D homonuclear COSY experiment for peracetylated p-D-glucose. 

A detailed descriphon of octanol-water distribuhon coefficient measurements by shake-flask can be found in publications by Dearden [2] and Hansch [24], The method usually involves the following solubilization of the compound in a mixture of mutually presaturated buffered water and octanol, agitation unhl equilibrium has been reached, careful separation of octanol and aqueous phases, and direct measurement of the solute concentration in both phases. Although seemingly simple, the method has a number of caveats making it inappropriate for some compounds. 

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. 

Because of their versatility and simplicity, TLC methods have been frequently applied to the separation and semi-quantitative determination of carotenoid pigments in synthetic mixtures and various biological matrices. The retention of pure carotenoid standards has been measured in different TLC systems. Separations have been carried out on silica plates using three mobile phases (1) petroleum ether-acetone, 6 4 v/v (2) petroleum ether-tert-butanol 8 2 v/v, and (3) methanol-benzene-ethyl acetate 5 75 20 v/v. Carotenoids were dissolved in benzene and applied to the plates. Developments were performed in presaturated normal chambers. The chemical structure and the Rv values of the analytes measured in the three mobile phases are listed in Table 2.1. It was concluded from the retention data that mobile phase 3 is the most suitable for the separation of this set of carotenoids [13],... 

The separation of synthetic red pigments has been optimized for HPTLC separation. The structures of the pigments are listed in Table 3.1. Separations were carried out on silica HPTLC plates in presaturated chambers. Three initial mobile-phase systems were applied for the optimization A = n-butanol-formic acid (100+1) B = ethyl acetate C = THF-water (9+1). The optimal ratios of mobile phases were 5.0 A, 5.0 B and 9.0 for the prisma model and 5.0 A, 7.2 B and 10.3 C for the simplex model. The parameters of equations describing the linear and nonlinear dependence of the retention on the composition of the mobile phase are compiled in Table 3.2. It was concluded from the results that both the prisma model and the simplex method are suitable for the optimization of the separation of these red pigments. Multivariate regression analysis indicated that the components of the mobile phase interact with each other [79],... 

The INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) experiment [6, 7] was the first broadband pulsed experiment for polarization transfer between heteronuclei, and has been extensively used for sensitivity enhancement and for spectral editing. For spectral editing purposes in carbon-13 NMR, more recent experiments such as DEPT, SEMUT [8] and their various enhancements [9] are usually preferable, but because of its brevity and simplicity INEPT remains the method of choice for many applications in sensitivity enhancement, and as a building block in complex pulse sequences with multiple polarization transfer steps. The potential utility of INEPT in inverse mode experiments, in which polarization is transferred from a low magnetogyric ratio nucleus to protons, was recognized quite early [10]. The principal advantage of polarization transfer over methods such as heteronuclear spin echo difference spectroscopy is the scope it offers for presaturation of the unwanted proton signals, which allows clean spec-... 

Here, presaturation is performed with the use of shaped pulses, which have a broader excitation profile. This method is therefore better suitable for the suppression of multiplets. The advantages of this technique are that it is easy to apply, easy to implement within most NMR experiments, and multiple presaturation is possible, and that it is very effective. The disadvantages are that transfer of saturation can occur (in aqueous solutions) to slowly exchanging protons that would be detectable without saturation. Another drawback is that spins with resonances close to the solvent frequency will also be saturated and 2D cross peaks will be absent. 

The choice of the suppression method to be used depends on both the solvent and sample characteristics. Therefore, HPLC-NMR suppression via presaturation... 

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... 

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). 

If desired, the methylmagnesium carbonate can be recovered as a white, brittle solid by evaporation of the solution under vacuum at temperatures up to 100°C. Unlike magnesium methoxide, which becomes almost completely insoluble on recovery as a solid, methylmagnesium carbonate solid is very soluble in methanol but tends to dissolve faster if the methanol is presaturated with carbon dioxide. The solid form provides a convenient method of storage or shipment of the material, much like instant coffee. 

All NMR spectra were collected on a Varian Unity 500 MHz spectrometer ( H frequency 499.8 MHz) equipped with a 5 mm inverse detection probe. Sample concentrations were typically 1 -2 mM and sample temperatures maintained at 25 C (unless otherwise noted). Sample pH was typically 3.5 - 4.0. Onedimensional H data were acquired with a H sweepwidth of 6000 Hz and an acquisition time of 2.3 seconds. The residual water signal was suppressed by presaturation. H DQF-COSY, NOESY and TOCSY (15) spectra were collected and processed using standard methods. All chemical shifts were referenced relative to internal DSS. 

To simplify the interpretation of STMAS spectra, it is desirable to remove the CT—>CT correlation signal, particularly when studying nuclei subjected to distribution of local environments or weak quadrupole interactions. Several methods are available to remove the CT CT peak. In the first method, the CT transition is presaturated by a soft pulse, prior to the STMAS sequence (Fig. 12a) [38]. A sequence consisting of a selective soft pulse followed by a delay and a short excitation pulse maybe used for optimising the presaturation pulse. Unfortunately, this simple method only works when aU species present in the sample have very similar transverse relaxation times and quadrupole couphng constants. 

Fig. 12a, b STMAS pulse sequences that suppress CT CT diagonal peaks using a a soft presaturation pulse b the subtraction method. In b the receiver phase is alternated between synchronised (bl) and non-synchronised (b2) for subtraction of the CT CT signal... 

Although commonly used in the past, presaturation is becoming much less popular for biomolecular applications mainly due to the advent of more efficient methods (as discussed below) and also due to a number of shortcomings such as those listed below ... 

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