Dead time stopped-flow mixing

For faster reactions the speed with which reactants can be mixed is a hmitation the mixing time must be less than the half-time. Stopped-flow techniques have been developed that permit remarkably short kinetic analysis times. Beckwith and Crouch described a stopped-flow kinetic analysis apparatus, with a mixing and dead time of less than 0.01 s, capable of analyzing 1000 phosphate samples per hour with a relative standard deviation of about 1%. Sample handUng, mixing, and gathering and evaluation of data were automated with the help of on-Une computer systems. 

Flow systems, both continuous and discrete, are used in kinetic-based determinations for monitoring fast reactions mainly. To this end (1) the dead time in the mixing system should be several orders of magnitude lower than the half-life of the reaction concerned and (2) nearly the whole kinetic curve must be recorded in order to implement reaction rate-based determinations and perform fundamental kinetic studies (e.g., the determination of reaction orders and rate constants). The advent of stopped-flow mixing and the continuous-addition-of-reagent technique has made noncatalytic reactions competitive with equilibrium methods in practical terms. 

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." ... 

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. 

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... 

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. 

High (linear) flow rates are reqnired to indnce turbulent mixing and to minimize the time between mixing and observation or quenching, known as the dead time . Due to the high flow rates, the pressure in the system increases. The working pressure of the mixing devices described above is maximally 10 bar (stopped-flow) and may be up to 20 bar in RFQ when viscous solutions are used. 

The combination of rapid mixing and fast detection systems allows cationic polymerisations to be followed on an even shorter time scale than with adiabatic calorimetry. Recent commercial stop-flow spectrophotometers have a dead time of about 15 msec, an improvement of more than one order of magnitude over previous home-made models. This implies that reactions with half lives of less than 100 msec can be analysed kinetically with a good degree of accuracy. Hi -vacuum techniques are not compatible with these instruments and all operations are therefore carried out in an inert atmosphere. 

Transient-kinetic techniques most often rely on the rapid mixing of reactants with enzyme to initiate the reaction. This mixing is essential so that all enzyme molecules start reaction in synchrony with one another therefore, the time dependence of the observable reactions dehnes the kinetics of interconversion of enzyme intermediate states. Because mixing requires a hnite amount of time, conventional methods are limited in their ability to measure very fast reactions. For example, a typical value for the dead time of a stopped-flow instrument is approximately 1 ms, which is because of the time it takes to hll the observation cell. Thus, reactions with a half-life of less than 1 ms (rate > 700 s ) are difficult to observe depending on the signal to noise... 

The limit for the measured rate constants is determined by the mixing rate and the instrument s dead time, defined as the time required for the solution to travel from the mixing chamber to the observation point. Nowadays, half-times in the millisecond range can be measured routinely. An extension of accessible rates up to 2000 s through algebraic corrections for mixing effects was discussed [11]. Under the assumption that the behavior of the solution at short times after mixing in the stopped-flow is described by the same equations that were found applicable for pulsed-accelerated flow, the precise rate constant can be obtained from a set of experiments carried out under pseudo-first-order conditions by use of Eq. 10. 

Dead time In column chromatography, the time, r, required for an unretained species to traverse the column in stopped-flow kinetics, the time between the mixing of reactants and the arrival of the mixture at the observation cell. 

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