NMR acquisition parameters

Figure 1 In vivo spectra acquired from the human occipital lobe at 4 T (top)and 7 T (bottom) using the same experimental setup and NMR acquisition parameters (TR=3 s and 128 signal averages). PE, phosphoethanolamine PC, phosphocholine Pi, inorganic phosphate GPE, glycerophosphoethanolamine GPC, glycerophosphocholine ...

Fully automated analysis is also an option wherein the samples are placed in an autosampler and predefined HPLC-NMR experiments are performed. This is covered in detail elsewhere in this volume, but in summary, the software allows automatic detection of UV peaks in the chromatogram based on predetermined time-windows or peak intensities. The successful detection of each UV peak triggers the system to stop the flow at an appropriate time to isolate the peak in the NMR flow probe. Then data relating to the peak (intensity, retention time, etc.) are transferred to the NMR host computer and used to define the parameters for the automatically acquired NMR spectrum. This automatic NMR operation includes field homogeneity optimisation, setting and optimisation of all NMR acquisition parameters, and the predefinition of the resultant signal-to-noise ratio required in the spectrum. The measurement of 2-dimensional (2D) NMR spectra can also be performed. 

Now let s look in detail at each process, so that we can understand the NMR acquisition parameters needed to set up the experiment. After the pulse, we will follow through the hardware devices in a block diagram and try to understand a little about the NMR hardware. It turns out that processing of the NMR signal in the hardware is strictly analogous to the theoretical steps we will use in viewing the NMR experiment with the vector model, so it is essential to understand it in general. 

Quantitative, 3C NMR has been used to determine the methoxyl contents of lignin in many wood species (Obst and Landucci 1986). The syringyl/guaiacyl composition in aspen MWL also has been measured using routine NMR acquisition parameters, but this method is restricted to carbons having the same chemical substituents and T, values and the results should be very carefully interpreted (Lapierre et al. 1984b). Semi-quantitative analysis can also be performed (Nimz et al. 1982). 

Due to these characteristics, the possibility to build databases of NMR-based metabolic profiles of foodstuffs, in order to assess their quality, is becoming more and more actual [9]. However, when we apply multivariate analysis to our data fields or wish to compare databases obtained by NMR spectroscopy by other researchers, it is very important to consider the source of extrinsic variability originated by the employed experimental conditions, in terms of sample preparation, NMR acquisition parameters, signal-to-noise ratio, NMR spectral pre-treatment, NMR data pre-processing, as well as the strategy for NMR-based metabolomics (for an exhaustive introduction to the reported critical cmicepts see the manual by Axelson [10]). 

A number of parameters have to be chosen when recording 2D NMR spectra (a) the pulse sequence to be used, which depends on the experiment required to be conducted, (b) the pulse lengths and the delays in the pulse sequence, (c) the spectral widths SW, and SW2 to be used for Fj and Fi, (d) the number of data points or time increments that define t, and t-i, (e) the number of transients for each value of t, (f) the relaxation delay between each set of pulses that allows an equilibrium state to be reached, and (g) the number of preparatory dummy transients (DS) per FID required for the establishment of the steady state for each FID. Table 3.1 summarizes some important acquisition parameters for 2D NMR experiments. 

Phase cycling The process of repeating a pulse sequence with identical acquisition parameters but with varying r.f. phase. This allows real NMR signals to add coherently whilst artifacts and unwanted NMR transitions cancel. 

One-dimensional proton NMR spectroscopy is the most straightforward method for process validation and development. It can be used as a limit test, i.e., to demonstrate that a particular analyte is below the detection limit. It can also be used to accurately quantify an analyte by comparing the NMR peak area from a test sample against a standard curve. To get accurate quantitation, it is important to keep the acquisition parameters and conditions constant for both standard and test samples. For example, the receiver gain, power level, and duration of all pulses must stay the same within an assay. In addition, the probe should remain tuned for all samples. 

Because sensitivity depends on so many different experimental factors, NMR spectroscopists generally use the signal-to-noise ratio, SIN, as a figure of merit for sensitivity comparisons. For example, in a comparison between NMR probes or spectrometers from two vendors, the spectral SIN measured for a standard sample acquired with specified acquisition parameters and probe geometry would provide a direct indication of relative sensitivity. The SIN is calculated for an NMR experiment as the peak signal divided by the root mean square (RMS) noise, given by Equation 7.6, and is directly related to the performance of the radiofrequency coil [3,6]... 

Reynolds FR, Enriquez RG, Choosing the best pulse sequences, acquisition parameters, postacquisition processing strategies, probes for natural product structure by NMR spectroscopy, JNat Prod 65 221—244, 2002. 

The type of the Fourier transformation depends upon a series of parameters AQ mod, FT mod and MC2, which are usually adjusted either before data acquisition or are automatically adjusted to suit the acquisition parameters. Nevertheless these parameters should be inspected in the General parameter setup dialog box and adjusted if necessary, before a 2D FT is initialized. For all the data available in the NMR data base, AQ mod in t2 is qsim (the two quadrature detected signals are sampled... 

Over the past 40 years fluorine nuclear magnetic resonance (19F-NMR) spectroscopy has become the most prominent instrumental method for structure elucidation of organofluorine compounds. Consequently the amount of spectral data published has grown almost exponentially Unfortunately NMR data for fluonnated compounds are not as well, or as easily, organized as proton data To facilitate retrieval of fluorine NMR information and comparison of data, acquisition parameters should be clearly defined Guidelines for publication of NMR data have been established by the International Union for Pure and Applied Chemistry (IUPAC) [7] The following niles for acquisition and reporting of NMR data should be strictly observed... 

As previously pointed out, the 33S NMR signal is often difficult to detect. Acquiring 33S spectra with an acceptable S/N may require from a few minutes to several days, depending on the symmetry of the sulphur electronic environment and molecular size. Therefore, a suitable choice of acquisition parameters and other experimental conditions (e.g. solvent and concentration in liquid phase, density in gas phase, temperature and so on) is particularly important. In addition, signal-processing methodologies can be critical for extracting all the information contained in a FID, especially when the S/N is not satisfactory. 

Unlike the sample condition, the experimental parameters have only a minor effect on the NMR spectral parameters. Experimental parameters such as spectral width, flip angle, repetition time, number of points in the free induction decay (FID) and in the real spectrum, number of scans, and processing parameters need to be comparable to those used for the acquisition of the database spectrum or spectrum of the authentic... 

The process of data acquisition results in an FID signal residing in the computer of the NMR instrument. In order to properly set up the acquisition parameters, it is helpful to understand a little about how this is accomplished. We will follow the sequence of events involved in the acquisition of the raw data for a simple ID ll spectrum on a 200-MHz instrument through a simplified diagram of the spectrometer ... 

At fast flow rates, the saturation of signals is avoided because spins leave the NMR active region, thus leading to an increase in S/N. The NMR linewidth is related to transverse relaxation time and, in a flow system, the linewidth is further influenced by the residence time. As a result, short residence times at fast flow rates yield broader NMR signals with low S/N. Ideally, detection of each analyte should be optimized using suitable acquisition parameters to obtain NMR spectra with comparable S/N. In addition, the overall temperature change in buffer by Joule heating may also contribute to linewidth increase [44],... 

Figure 14 Stopped-flow TOCSY CEC-NMR spectra of paracetamol glucuronide. Acquisition parameters number of 144 scans for each of 256 increments. Spectral width of 4716 Hz, number of points 4096. Processing parameters zero filled and multiplied by apodization function of 3 Hz in both dimensions. (From Ref. 52 reproduced with permission from The Royal Society of Chemistry.)...

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