ID NMR Experiments

Four general classes of NMR experiments are routinely used to analyze metabolites (1) ID NMR experiments (2) 2D NMR experiments (3) Solvent suppression methods and (4) Hyphenated NMR experiments. The ID and 2D NMR experiments are commonly used for metabolite structure determination. The various solvent suppression techniques (Gaggelli and Valensin, 1993 Hwang and Shaka, 1995 Smallcombe and Patt, 1995) are crucial for dilute metabolite samples where the solvent peak is the most intense peak in the NMR spectrum. These solvent suppression techniques can be incorporated as needed in both ID and 2D NMR experiments. Since their introduction in the 1990s, hyphenated NMR methods have become common tools in the identification of metabolites. These methods include LC-NMR (Albert, 1995 Spraul et al., 1993, 1994), LC-NMR-MS (Mass Spectrometry) (Shockcor et al., 1996) and LC/SPE (solid phase extraction)/NMR (Alexander et al., 2006 Bieri et al., 2006 Xu et al., 2005 Wilson et al., 2006). 

In general, both ID H and NMR spectra are collected to resolve a metabolites structure. ID NMR spectra of other heteronuclei ( N, F, P) 

A ID nuclear overhauser experiment (NOE) (Gaggelli and Valensin, 1993) is an important and valuable variation on the simple ID NMR experiment that provides spatial relationship between each nucleus in the structure. Coupling constants observed in a ID H NMR spectra provide connectivity for directly bonded nuclei whereas, a ID NOE experiment identifies nuclei that are close in space ( 6A). Briefly, a ID NOE experiment requires the addition of a second lowpowered rf pulse that selectively saturates a specific peak in the NMR spectrum. The saturated peak becomes a null in the spectrum and any other nuclei that are coupled through space via a dipole-dipole interaction to the saturated peak will experience a change in peak intensity. A ID NOE experiment requires collecting two NMR spectra, with and without saturation, to monitor changes in peak intensity. A summary of common ID NMR experiments and their applications are listed in Table 12.6. 

A fundamental component of the interpretation of NMR data is deciphering the NMR assignments, which correlates an observable NMR resonance with a specific atom in the molecular structure of the metabolite. This process is illustrated using the structure and H NMR spectrum of 1,3-dimethylnaphtha-lene as an example (Fig. 12.6). The two methyl groups have distinct H NMR chemical shifts because of their unique local environments. The NMR assignment process results in attributing the NMR peak at 2.57 ppm to methyl (a) and NMR peak 2.39 ppm to methyl (b). 

Verify or eliminate proposed structures by comparing the changes in chemical shifts, coupling constants, and integrations with that of the parent molecule. 

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Why are we much more likely to have signals outside the spectral width (SW) in an average 2D NMR experiment than in a ID NMR experiment Why do spectral widths in a 2D NMR need to be defined very carefully, and what effects will this have on the spectrum ... 

Jeener s idea was to introduce an incremented time ti into the basic ID NMR pulse sequence and to record a series of experiments at different values of second dimension to NMR spectroscopy. Jeener described a novel experiment in which a coupled spin system is excited by a sequence of two pulses separated by a variable time interval <]. During these variable intervals, the spin system is allowed to evolve to different extents. This variable time is therefore termed the evolution time. The insertion of a variable time period between two pulses represents the prime feature distinguishing 2D NMR experiments from ID NMR experiments. 

Apart from the separation into two fractions, the propagator can also be interpreted in terms of an average quantity, the second moment of displacements, which is proportional to the dispersion coefficient D (A). Rather than computing D ( A) from the shape of the propagator directly, it is also possible to obtain it from the initial slope of the signal function E(q,A) in a ID NMR experiment [43] ... 

ID NMR experiments with multiple polarization transfer steps... 

ID NMR experiments are often used for quality-control purposes and can readily be used to confirm the purity of peptides. Simple ID spectra are also often used to determine whether a peptide is structured or unstructured or whether aggregation is present. Dispersion of the amide chemical shifts is an indicator of the former, whereas a narrow distribution, in the range of 7.5-8.5 ppm, is characteristic of unstructured peptides. Aggregation leads to broad peaks and spectra of poor quality. Adjustment of conditions by varying pH, buffer, cosolvent (e.g., acetonitrile for hydrophobic peptides), or peptide concentration and monitoring the effects on ID spectra is often used to find optimum conditions. 

This traditional ID NMR experiment involves selectively decoupling a proton from its J coupling partners. This is accomplished by low-power irradiation at the frequency of the... 

A ID NMR experiment provides information on the chemical shift and spin-spin coupling fine structure of the individual resonances in the spectrum. Double or multiple pulse irradiation experiments provide additional data on through bond scalar connectivities or through space dipolar connectivities, which relate to resonance assignments, conformational state and dynamics of the molecules under investigation. 

Figure 4.24. Schematic representation of the difference in glass connectivity information obtainable from ID NMR experiments (small observation window) and 2D experiments (larger window) for A, B. homogeneous glasses, C, D. glasses containing regions of inhomogeneity.

Data acquisition is approached very differently in 2D, compared with ID, NMR experiments. The reason is that we are now dealing with, at minimum, a one-dimensional data matrix (for 2D NMR) and, perhaps, two or three matrices (for 3D and 4D NMR, respectively) for complex biological molecules,... 

Like acquisition, data processing is performed differently in 2D, compared with ID, NMR experiments. The principal reason is that signal truncation is a much more serious problem in 2D than ID experiments. Zero filling also is used in 2D experiments, as is the relatively new technique of linear prediction. 

While many NMR active nuclei such as JH, 13C, 31P, and 15N have been used to analyze metabolites (19-21), JH NMR analysis is the most widely used in the field because of the ubiquitous nature of JH and its high NMR sensitivity. Furthermore, ease of analysis and high-throughput capabilities make onedimensional (ID) NMR experiments, including the ID NOESY (nuclear Over-hauser enhancement spectroscopy) and CPMG (Carr-Purcell-Meiboom-Gill)... 

Parella and Bellow have described both non-selective and selective versions of several proton-detected ID NMR experiments that can be applied to Using these methods, the authors reported the successful determination of both one-bond and long-range coupling constants using a model tripeptide at natural abundance. 

The various ID CP NMR spectra of a biaxially drawn industrial PET film are shown in Fig. 14.7. The pronounced sensitivity to the orientation of the sample with respect to the applied magnetic field is also shown [7]. However, the orientation distribution is relatively complicated and, therefore, difficult to quantify from these data. For PET, overlapping resonances from carboxyl and phenylene group carbons are especially troublesome and, consequently, restrict the angular information achievable in ID NMR experiments. 

Fig. 1 The half-lives of amide proton-deuterium exchange of amides la-c based on ID NMR experiments (500 MHz, CDC13 DMSO-d7 D20 = 2 19 19)...

The idea of two-dimensional (2D) NMR spectroscopy was introduced in 1971 by Jeener, and several experimental demonstrations were soon reported by Ernst and co-workers. Since then many new 2D NMR experiments have been designed to give higher resolution of the resonances and information about the spectral parameters, and therefore the structural details, that would be inaccessible or at least more laborious to determine with ID NMR experiments. The improved resolution in 2D NMR experiments has paved the way for general acceptance that NMR spectroscopy is a valid analytical technique in analysis of complex samples. ... 

Use of quantitative 2D NMR spectroscopy is still quite rare in food analysis and quality control. A possible reason can be that experimental set-up, acquisition and processing of 2D NMR experiments are considered to be too difficult or time-consuming compared to ID NMR experiments. Also, restrictions and regulations from the laboratory environment (quality systems, standards) can hinder application of new methodology in quantification. Considering the challenging sample matrices the 2D NMR methods would be excellent tools in quantification. 

Figure 5.11 Alternative diagrammatic representation of general FT ID NMR experiment. There is a single 90° pulse then signal observation and acquisition in time domain tj prior to fourier series transformation of time domain signal information SFiD(ti) into frequency domain (spectral intensity) information, 7nmr(Fi).

Limited to compounds with on column loadings >10 jjig Limited to ID NMR experiments.Broadening of the NMR signal due to on-flow conditions... 

The simplest ID NMR experiments involve the apphcation of a pulse followed by observation of the resulting signal in the time domain, with subsequent Fourier transformation of the data to the frequency domain for presentation in a format that we, as chemists, can understand. Pulsed NMR methods had their inception in 1966 [33] and have almost completely supplanted earlier continuous wave (CW) methods. For reasons of sensitivity, only ID NMR spectra were typically acquired prior to the 1970s. The advent of pulsed Fourier transform NMR instruments made it possible to acquire natural abundance C NMR spectra on a routine basis in the early 1970s. With the routine availabihty of C NMR data came the compilation of chemical shift data bases and a very different way of approaching chemical structure elucidation. 

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