Instrument time

NMR spectroscopy is always struggling for increased sensitivity and resolution, as well as more efficient use of the instrument time. To this end, numerous improvements of the simple inversion-recovery method have been proposed over the years. An early and unportant modification is the so-called fast mversion recovery... 

Assuming that the instrument response is first order, then as shown in Sec. 2.1.1.1, the instrument time constant Xm is then given by the value of time at the 63% point (response to a step-change disturbance), where... 

This was probably the most difficult chapter to put together in this book. For many people who use NMR spectrometers, there will be little (or no) choice about parameters for acquisition - they will probably have been set up by a specialist to offer a good compromise between data quality and amount of instrument time used. This could make this chapter irrelevant (in which case you are welcome to skip it). But if you do have some control over the acquisition and/or processing parameters, then there are some useful hints here. This brings us on to the next challenge for the section - hardware (and software) differences. You may operate a Bruker, Varian, Jeol or even another make of NMR spectrometer and each of these will have their own language to describe key parameters. We will attempt to be vendor neutral in our discussions and hopefully you will be able to translate to your own instrument s language. 

As in the case of all NMR problem-solving, the issue is always one of using the most appropriate tool for the job. The two techniques are in no way mutually exclusive. Too much data is not a bad thing if the instrument time is available but taking a chance on insufficient data can be a costly mistake in the long run. 

We have tried to point you in the direction of the experiments that we have come to use and rely on, with good reason. There are dozens more out there that have been developed some have evolved and are now generally known by another name (e.g., the ROESY experiment used to be known as CAMELSPIN) and some have been superseded and fallen by the wayside. If you have the instrument time and the inclination, by all means play but if time is of the essence, as it usually is, stick with the safe options. 

In practical applications, it can be a factor that the above approach by virtue of the cycle over A values has a higher minimum number of scans per ti value than the standard experiment and its various accordion versions. For dilute samples, this does not matter but for concentrated samples the instrument time can be longer than required considering the inherent sensitivity. 

NMR techniques are unique in their ability to resolve internal dynamics with site-specific probes. Backbone dynamics may be derived from relaxation data of 15N nuclei. Relaxation data are conveniently measured in experiments that utilize [15N,1H]-HSQC-derived pulse sequences and hence can be performed within less than a week of total instrument time with a 1 mM sample (at one field). The underlying principle of the measurement is described in Chapt. 12 and has also been recently reviewed by Palmer [91]. 

For accurate determination of protein molecular weight, mass spectrometry and LC-MS have largely displaced SDS-PAGE. However, SDS-PAGE will still be used where estimates of molecular weight suffice or where MS instrument time is limited. 

At this point, it is worthwhile to return on the theoretical basis of the kinetic method, and make some considerations on the assumptions made, in order to better investigate the validity of the information provided by the method. In particular some words have to been spent on the effective temperamre The use of effective parameters is common in chemistry. This usually implies that one wishes to use the form of an established equation under conditions when it is not strictly valid. The effective parameter is always an empirical value, closely related to and defined by the equation one wishes to approximate. Clearly, is not a thermodynamic quantity reflecting a Maxwell-Boltzmann distribution of energies. Rather, represents only a small fraction of the complexes generated that happen to dissociate during the instrumental time window (which can vary from apparatus to apparatus). 

As a consequence a quantitative C NMR spectrom calls for 5-10 times the instrument time of a purely qualitative one. It is not feasible to increase the concentration of the polymer in solution excessively because this involves an increase in linewidth and the gain in time (considering the lower number of transients required) is accompanied by a poorer quality of the spectrum. The planning of a high quality C NMR experiment requires a fine balance between results, time, and costs. 

This work has been supported by the Swedish Natural Science Research Council. The grant of the U300 instrument time at the Swedish NMR Center is gratefully acknowledged. 

Summing up, one can say that, in the experience of the authors, an organic sample of about 400 Da molecular weight and a solubility of 100-200 mg in 3 ml of solvent requires no more than 3 hours of instrument time, usually much less. Biological samples, unfortunately, often do not meet these requirements and, subsequently, studies on these subjects are more rare, as is shown also in Section m of this chapter. 

The major advantage of this dramatic time reduction lies in the ability to scan the sample repeatedly, combine the relaxation-decay patterns collected from each scan, and then perform a Fourier transform upon the final composite relaxation-decay pattern. This technique, in essence, increases the spectral sensitivity by allowing the NMR signals acquired from each scan to be constructively added to each other while the noise cancels itself de-con structively. This approach greatly increases the sensitivity of the instrument and allows NMR experiments to be performed on samples that have low concentrations of the desired nucleus (i.e., for 31P, 20 mg of P/L is a feasible concentration with instrument time of hours to a few days). 

There is reasonably good agreement between the measurements made by spectral reconstruction and the linear wavelength method. This supports the validity of this method which takes considerably less instrument time (see above). 

Setting up a fixed number of transitions either wastes instrument time if peaks are more concentrated than expected or leads to a complete loss of results for lower-concentration peaks. A sensible procedure which measures the spectrum up to a predefined signal-to-noise level, or even stops the acquisition if the sample concentration is too low, helps dramatically to save instrument time. 

Needs for improved measurement methods differ depending on whether one is considering low or high transmission rate materials. In the former case one needs very sensitive detectors. Selectivity is also desirable so that interferences from extraneous species can be avoided. In the case of high transmission rate materials instrumental time constants and saturation effects need to be better understood. In all cases there is a need for more convenient instruments and a better knowledge of their operating principles. 

Combinatorial Mixture Screening The increased popularity of LC/MS-based methods combined with limited resources resulted in advances that effectively matched combinatorial chemistry samples (i.e., complexity) with instrument time. Richmond, Yates, and coworkers (Richmond et al, 1999 Yates et al.,2001) demonstrated the use of flow injection analysis (FIA)-LC/MS systems for rapid purity assessment and combinatorial mixture screening, respectively. These LC/MS-based applications addressed two critical bottlenecks HPLC... 

Greater efficiency (sample preparation time, run set-up time, instrument time). 

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