Methods of Obtaining NMR Spectra

FIGURE 2.8 Formation of the effective rf field Brii in a frame rotating at o radians/second. Beff is the vector sum of applied rf field B, along x and the residual field along z resulting from B0 and the fictitious field that represents the effect of the rotating frame. 

We found in Section 2.7 that the fundamental process for obtaining an rf NMR signal requires tipping the magnetization M away from the z axis to create a precessing component in the xy plane. We now inquire a little more closely into the means by which a set of magnetizations of differing Larmor frequency (e.g., a set of nuclei of different chemical shifts) can be so tipped. 

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I assume that you are conversant with basic principles of XH or proton NMR spectroscopy as applied to small molecules. In particular, I assume that you understand the concepts of chemical shift (8) and spin-spin coupling, classical continuous-wave methods of obtaining NMR spectra, and decoupling experiments to determine pairs of coupled nuclei. If these ideas are unfamiliar to you, you may wish to review NMR spectroscopy in an introductory organic chemistry textbook before reading further. 

The modem methods of taking NMR spectra involve the use of very short radio frequency pulses (of variable duration from 1 to 200 ms) instead of a continuous signal as in older NMR. This requires full automation of the test, the Fourier transform analysis, data storage and multiple scan capability. With the scalar (low power, ca. 4 kHz) and dipolar (about 45 kHz) decoupling, magic angle spinning and cross polarization methods one can obtain spectra of solid samples with resolution similar to those known for liquids. The spectra provide precise information on the... 

Not only is pulsed FT NMR the best method for obtaining proton spectra it is the only practical method for many other nuclei including It also makes possible a large number of sophisticated techniques that have revolutionized NMR spectroscopy... 

To obtain statistically significant comparisons of ordered and disordered sequences, much larger datasets were needed. To this end, disordered regions of proteins or wholly disordered proteins were identified by literature searches to find examples with structural characterizations that employed one or more of the following methods (1) X-ray crystallography, where absence of coordinates indicates a region of disorder (2) nuclear magnetic resonance (NMR), where several different features of the NMR spectra have been used to identify disorder and (3) circular dichroism (CD) spectroscopy, where whole-protein disorder is identified by a random coil-type CD spectrum. 

FIGURE 44. Tilt series of 2H NMR spectra from the deuteriomethyl groups Cl 8 (left column) and C20 (right column) of retinal in oriented purple membranes, at seven different sample inclination (a) in the spectrometer field. The line-shape simulations are superimposed over the experimental spectra in order to illustrate the good line fit obtained by the prediction method. Reprinted with permission from Reference 57. Copyright (1994) American Chemical Society... 

Similar spectra can be obtained more rapidly and with less sample if the data are acquired through the proton signals, which are much more intense. Basically, the H NMR data are acquired and the H- C coupling constant used as the delay in a pulse sequence, which enables us to obtain the carbon spectrum. This method of obtaining the data is called inverse-mode , since the carbon atoms are detected through their attached hydrogen atoms rather than by direct detection, with obvious benefits in the sensitivity and the time taken to obtain a spectrum. HMQC and HMBC are both examples of inverse-mode spectra and this method is so much quicker than CH COSY that an entire HMQC spectrum can be obtained in much less time than it takes to obtain the proton-decoupled C... 

Most published 15N chemical shifts have been measured using 15N-labelled compounds alternatively, a long-term accumulation of 15N NMR spectra at the natural abundance level of the 15N nucleus had to be used. H inverse-detected NMR spectra of 15N-labelled and nonlabelled l-phenylazo-2-naphthol were measured to test the applicability of this method and to determine the concentrations of dye necessary to obtaining NMR signals.78... 

From the results in Table 6, the differences between NMR-based and probabilistic model-based estimates of carbonyl -f carboxyl , aromatic , and alkoxy - - alkyl are —8%, —5%, and - -14% for FAs, —11%, —3%, and -K3% for HAs, and —2%, - -1%, and 0% for NOM. The discrepancies observed for most forms of carbon in FAs and HAs are greater than 5%, and they are consistent with theory and observation for CP-MAS NMR spectrometry, in which the ratio of ip -hybridized carbon to ip -hybridized carbon is significantly underestimated (Mao et al., 2000). For NOM, the two approaches yield highly consistent estimates of the distribution of organic carbon. It is perhaps significant that most of the NMR spectra for NOM samples in Figure 7 were obtained on aqueous solutions, not on solid samples, i.e., the CP-MAS method was not used. 

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