Stopped-flow experiments time resolution

The time resolution of stopped flow experiments is typically 1-2 ms,21 and is determined by the time required to mix the solutions, flow the mixed solution to the detection chamber, and stop the flow. Smaller detection cells can be used to decrease the time resolution at the expense of the signal-to-noise ratio of the detected signals. Various kinetic traces have to be averaged to achieve good kinetic profiles and sample volumes of milliliters with concentrations of micromolar to millimolar are required. 

A stopped-flow experiment is quite simple in principle. The apparatus allows the rapid mixing of two or more solutions, which then flow into an observation cell while the previous contents are flushed and replaced with freshly mixed reactants (Fig. 1). A stop syringe is used to limit the volume of solution expended with each measurement and also serves to abruptly stop the flow and to trigger simultaneously a computer to start data collection. Thus, if one were to watch from the point of view of the photodetector, one would first see the solution flow into the observation cell and then abruptly stop. The reaction is followed as the solution ages after the flow stops. The time resolution of the method... 

If the retention times of the analytes are known, or there is an efficient method for their detection on-line, such as UV, MS or radioactivity, stop-flow HPLC-NMR becomes a viable option. In the stop-flow technique, all the usual techniques available for high-resolution NMR spectroscopy can be used. In particular, these include valuable techniques for structure determination such as 2-dimensional NMR experiments which provide correlation between NMR resonances based on mutual spin-spin coupling such as the well-known COSY or TOCSY techniques. In practice, it is possible to acquire NMR data on a number of peaks in a chromatogram by using a series of stops during elution without on-column diffusion causing an unacceptable loss of chromatographic resolution. 

Kinetic experiments are performed in two different ways. In one an initial disequilibrinm exists between two or more reactants, which after being rapidly mixed, combine to react toward equilibrium see Rapid Scan, Stopped-Flow Kinetics). Ideally, the mixing time is short with respect to the timescale of the reaction or actually with respect to the formation of intermediates. In contrast, in the relaxation experiment, the reactants are together and in equilibrium, and the whole system is instantaneously displaced from equilibrium. Subsequently, the system relaxes to the same or a new equilibrium state. Table 1 suimnarizes the approximate time resolution of various commonly applied mixing and relaxation techniques. The table indicates the superiority of the relaxation methods with respect to time resolution, mainly due to the development of ultrafast lasers. Mixing liquids on the (sub)microsecond time scale appears to present an important experimental barrier. 

The decision to use LC/NMR involves a decision also as to the mode of NMR detection to be used in the analysis. The alternatives are stopped flow, loop capture, or continuous flow. The latter is probably the most obvious use of LC/ NMR to a chromatographer - the NMR becomes a detector in the way that a photodiode array detector or MS might be used. Structural information will obviously be available from this use but typically the constraints of continuous flow preclude the structural analysis of low-level components. For example, in a typical continuous flow experiment at ImLmin", the need is to acquire as many NMR spectra per unit time as is possible to give sufficient S/N in the NMR spectrum while retaining adequate time resolution in the chromatographic dimension. Thus 32 scans may be acquired per time increment, resulting in... 

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