Stopped-flow dead time

When solutions of [Pd(H 2GGG)] are mixed with acid, there is an initial, rapid absorbance change (A i) complete within the stopped-flow dead-time. The three subsequent first-order reactions (k, k, are separated in rate by only about one... 

Rgure 4 Analysis of time course to measure the flow time and the dead time. The first 10 ms of the traces In Figure 3 are shown as individual data points. The solid lines are the exponential fits to the data extrapolated back to true time zero. The flow period is the flat initial portion of the curve and, in principle, may have any length and depends on the time between triggering data capture and the flow stopping. The dead time is the time between t = 0, given by the point of intersection of the cun/es, and when flow stops. In this experiment the dead time and flow period are approximately the same, but need not be so. 

The dead time is typically 3-5 ms. so stopped flow is not quite as fast as continuous flow, but it requires less than a milliliter of each solution per run. Methods have been described for measuring the dead time " " these are based upon standard reactions whose kinetic behavior is well known. The error introduced by collecting data before mixing is complete can be corrected." ... 

Stopped-flow kinetics. If one uses an apparatus with a dead time of 2.3 ms, what fraction of a second-order reaction is missed if the initial concentrations are 2.0 X 10 3 M and 6.8 x 10 3 M, given a rate constant of 3.7 x 103Lmol 1 s-1 ... 

The influence of inhibitor on the performance of a semi-continuous reactor can be, in some ways, similar to both batch and continuous systems. A dead time is usually observed upon addition of the initial charge. When the secondary stream flow is started after some reaction of the initial charge, additional inhibitor flows into the reactor and the initiation rate drops. When this programmed addition is stopped the initiation rate increases sometimes enough to cause temperature control problems. 

Stopped-flow CD studies of protein folding have generally revealed a species that forms within the dead-time of the experiment, 10 ms (Kuwajima et al., 1985, 1987). The 222 nm ellipticity of this so-called burst-phase intermediate is generally substantially more negative than... 

The burst phase amplitude represents the percentage change in the far-UV CD signal (216-225 nm) occurring in the dead time of the stopped-flow experiment relative to the total change on folding. 

Reactions which cannot be perturbed by changing an external parameter may be detected by the stopped-flow method. The detection system of this apparatus is the same as that of the pressure-jump apparatus described previously (10). For this system, aqueous electrolyte solution and an aqueous metal-oxide suspension are mixed rapidly by operating an electric solenoid valve under nitrogen gas of 7 atm. The dead time of this apparatus is 15 ms. 

When a larger than tenfold excess of superoxide is applied to the solution of [Fe Por)(DMSO)2l, the reduced [Fe (Por)] complex is formed during the dead time of the stopped-flow instrument, and the second reaction step (Scheme 12), i.e., formation of the peroxo [Fe (Por)(02 )] complex is observed (Fig. 10). This second... 

The thematic approach to isolating the deacylation step is to generate the acylen-zyme in situ in the stopped-flow spectrophotometer by mixing a substrate that acylates very rapidly with an excess or stoichiometric amount of the enzyme. The acylenzyme is formed in a rapid step that consumes all the substrate. This is then followed by relatively slow hydrolysis under single-turnover conditions. For example, acetyl-L-phenylalanine p-nitrophenyl ester may be mixed with chy-motrypsin in a stopped-flow spectrophotometer in which the enzyme is acylated in the dead time. The subsequent deacylation may be monitored by the binding of proflavin to the free enzyme as it is produced in the reaction.8... 

The stopped-flow method is a routine laboratory tool, whereas the continuous-flow apparatus is used in a few specialized cases only. The stopped-flow technique requires only 100 to 400 /XL of solution or less for the complete time course of a reaction the dead time is as low as 0.5 ms or so and observations may be extended to several minutes. Stopped flow does, however, require a rapid detection and recording system. 

Bio-Logic Instrument and Laboratories (Meylan, France) manufactures an SFM-3 stopped-flow instrument (Fig. 4.17) that consists of three independent drive syringes driven by stepping motors, two mixers and a delay line, three observation windows, replaceable cuvettes, no stop-syringes, and efficient temperature regulation. At maximum flow rate, the minimum dead times range from 1.0 to 4.9 ms for fluorescence detection and 1.3 ms for transmittance. Currently, the Bio-Logic MOS-IOOO optical system employs fluorescence or absorbance detection, which is not suitable... 

The measurement of fast chemical reactions at the liquid-liquid interface is very difficult, since the reaction which starts just after the contact of two phases has to be measured. The dead time of the HSS method was several seconds and that of the two-phase stopped flow method was from few tenth millisecond to several hundred milliseconds. The micro-two-phase sheath flow method is the only method that can measure such fast interfacial reactions, which finish within 100 ps. An inner organic microflow was generated in an outer aqueous phase... 

Reactions completed within the dead time of stopped-flow instruments cannot be measured dead times are on the order of milliseconds for conventional instruments but can be somewhat shorter if special mixers are used. 

Because stopped-flow techniques are widely used with optical detection, samples should be prepared in solution and produce detectable signal changes after mixing into the cell. In some situations, if the reaction of some samples is very rapid and complete within the dead time of the stopped-flow instruments, the majority and indeed the entire kinetic time course may be lost. Selected adjustment of concentration, solution conditions, temperature, and so on, may be able to slow the reaction into an accessible time range, but this is not always possible or desirable. Such systems are not amenable to the stopped-flow technique. In general, other techniques will have to be used, and these will be... 

It is good practice to check from time to time that the stability of the instrument is within the manufacturer s specification. It is necessary to test the reliability of the stopped-flow instrument using control experiments that test a range of parameters such as the dead time, mixing efficiency and signal output. In general, these tests will be the same for the instrument in the configuration for fluorescence studies as that for absorbance studies. 

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