Stopped-flow time resolution

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... 

During the course of these studies the necessity arose to study ever-faster reactions in order to ascertain their elementary nature. It became clear that the mixing of reactants was a major limitation in the study of fast elementary reactions. Fast mixing had reached its high point with the development of the accelerated and stopped-flow teclmiques [4, 5], reaching effective time resolutions in the millisecond range. Faster reactions were then frequently called inuneasurably fast reactions [ ]. 

Advantages. Polarization measurements permit continuous binding analysis with subsecond resolution if required. When applied in stop-flow mixing conditions the technique has the best time resolution of the methods presently available. 

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. 

As might be expected, the problem of obtaining spectra of a reacting system increases as the time resolution involved decreases. The spectral changes associated with a reaction may be constructed by wavelength point-by-point measurements. The method, although tedious and costly on materials, is still used. However rapid-scan spectrophotometry, linked to stopped-flow, is now more readily available and reliable. Two systems are used, shown schematically in (3.29) and (3.30). An example of its use is shown in Fig. 3.9. Rapid scan... 

The chromatographic stage is not interrupted and therefore no stop-start effects will create disturbances. The peaks are separated in the storage loops, and therefore the NMR measurement time is not limited and will not decrease the performance of other peaks. In complex chromatograms the chance of finding the peak(s) of interest is dramatically increased. As in the direct stop-flow mode, the static conditions provide stability and the best NMR conditions for the acquisition of all kinds of high-resolution ID and 2D NMR spectra. 

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. 

If the retention times of the compounds to be separated are known, or if they can be detected by using UV (including diode arrays), radiochemical or fluorescence detectors, stop-flow LC-NMR becomes an option. Upon detection, the PC controlling the liquid chromatograph allows the pumps to continue running, moving the peak of interest into the NMR probe. Once the pumps have stopped, normal high-resolution NMR spectroscopy is possible. It could be... 

Comparison of the stop-flow spectra from ethylbenzene in the liquid and in the supercritical state, recorded at a temperature of 323 K and a pressure of 165 bar, shows that there is no degradation in resolution in going from the liquid to the supercritical state (Figure 7.2.9). Because the NMR signal line widths have a reciprocal relationship to the spin-spin relaxation time T2, it is evident that T2 for protons does not change dramatically in the supercritical state. However, this is not the case with respect to the spin-lattice relaxation time T. ... 

M 35] [protocol see [119]] A protein conformation kinetic study of the small protein ubiquitin was performed both in the continuous and in a stopped-flow mode at low reactant consumption [119], The bifurcation mixer was used prior to an IR flow cell for data monitoring. The change of conformation from native to the A-state was followed when adding methanol under low pH conditions to the protein solution. In the continuous mode, long data acquisition could be made and the reaction time was determined by the flow rate and the volume interconnecting zone between the mixer and IR flow cell, which was small, but not negligible. In the stopped-flow mode, the reaction time resolved was dependent on the time resolution of the FTIR instrument. 

M 91] [P 83] The mixing device was tested as a measuring tool for studying fast consecutive reactions and processes [6]. This concerned applications in quench-flow and stopped-flow analysis, where a time resolution of < 1 ms is required. 

We compared the l3C NMR spectra of the subtilisin complexes of [M]SSI and [M]SSI. Rather surprisingly, the NMR spectrum taken within two hours after the preparation of [M]SSI -subtilisin BPN complex was absolutely identical to that of [M]SSI-subtilisin BPN. A period of two hours was necessary to obtain the 13C NMR spectrum of the [M]SSI -subtilisin complex with a sufficient signal-to-noise ratio. This included the time after the modified inhibitor was brought in contact with subtilisin BPN. There were no extra signgals detected other than those observed for the [M]SSI-subtilisin complex, indicating that the cleaved scissile bond in [M]SSI can be rapidly restored in the complex. Since time-resolution of 13C NMR spectroscopy is rather limited by its inherent insensitivity, we are not able to tell exactly how fast this process is. Tonomura et al., however, have recently found by a stopped flow technique an unknown kinetic process having a half-life time of two seconds for the SSI -subtilisin system. Obviously this process should be the restoration process of the cleaved scissile bond of SSI in the complex. Therefore, the hydrolyzed scissile bond could in fact be restored within several seconds (private communication). 

In particular the higher time resolution compared to the stopped-flow ( 1 ms) is important in this respect. In enzymes, many intermediates are formed on the microsecond time scale but have so far escaped detection. 

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. 

A major breakthrongh in time resolution ( 1 ms), dynamic range ( unlimited ) and reduction of sample amount came with the development by Britton Chance of the stopped-flow techniqne see Rapid Scan, Stopped-Flow Kinetics), which is stUl widely used today. The stopped-flow techniques finds a major application in the... 

Structural Aspects of Microemulsions. Several investigators have studied the structure of microemulsions using various techniques such as ultracentrifugation, high resolution NMR, spin-spin relaxation time, ultrasonic absorption, p-jump, T-jump, stopped-flow, electrical resistance and viscosity measurements (56-58). The useful compilation of different studies on this subject is found in the books by Robb (68) and Shah and Schechter (69). Several structural models of microemulsions have been proposed and we will discuss only a few important studies here. 

On-flow HPLC-NMR analysis can also be performed when sufficient material is available. It involves collecting the NMR data continuously as the sample passes through the probe. This is the most efficient method for stmcture evaluation by HPLC-NMR. The NMR data are represented in a 2-D plot where the x direction contains chemical shift information and they direction is representative of the LC retention time. The individual spectra can be extracted from the ID slices along the x axis if so desired. The resolutions in the individual spectra are of somewhat lower quality than in the stop-flow method however, the introduction of the second dimension allows for easy stmcture assignment even for overlapping peaks in the LC separation. As seen in Fig. 19, the on-flow HPLC-NMR characterization shows four distinct sets of resonances. 

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