Continuous-Flow Mixing Techniques

The parameter y ax can be converted to a turnover number or kcat by taking account of the molar concentration of enzyme present in the assay mixture. The values of kcat of enzymes are generally in the range of 10 -10 12] means that the catalytic events on the enzyme occur in the time range of milliseconds or less. In order to characterize such steps, it is necessary to employ rapid reaction techniques such as stopped-flow, which has a dead time of approximately 1 ms. Continuous-flow mixing techniques can have shorter dead times but make much greater demands in terms of quantities of sample required. The value of kcJK provides not only a quantitative measure of the specificity of an enzyme for a given substrate but it can also be used as a measure of catalytic efficiency... 

There are many potential advantages to kinetic methods of analysis, perhaps the most important of which is the ability to use chemical reactions that are slow to reach equilibrium. In this chapter we examine three techniques that rely on measurements made while the analytical system is under kinetic rather than thermodynamic control chemical kinetic techniques, in which the rate of a chemical reaction is measured radiochemical techniques, in which a radioactive element s rate of nuclear decay is measured and flow injection analysis, in which the analyte is injected into a continuously flowing carrier stream, where its mixing and reaction with reagents in the stream are controlled by the kinetic processes of convection and diffusion. 

Two techniques conceptually related to classical continuous flow make use of different injection methods. In one of these a reactant solution formed into a highspeed jet is injected through a sheet or film of the second solution. The jet speed is 40 ms , and the mixing time is 1 p.s. 

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. 

This section deals briefly with classical methods based on conventional mixing of the sample and reagents such as the batch mode and low-pressure flow mixing methods, as well as the use of CL detection in continuous separation techniques such as liquid chromatography and capillary electrophoresis for comparison with the unconventional mixing mode. 

In this technique, which was developed in the 1970s, microlitre volumes of liquid sample are injected, at intervals, into a continuously flowing carrier stream which is not air-segmented. Various reagent streams are introduced as required and controlled mixing of reagents and sample occurs. The fact that flow injection analysis does not involve air-segmented streams makes it possible to include such separation steps as solvent extraction and gas diffusion. 

Solvent extraction can be automated in continuous-flow analysis. For both conventional AutoAnalyzer and flow-injection techniques, analytical methods have been devised incorporating a solvent extraction step. In these methods, a peristaltic pump dehvers the hquid streams, and these are mixed in a mixing coil, often filled with glass ballotini the phases are subsequently separated in a simple separator which allows the aqueous and organic phases to stratify. One or both of these phases can then be resampled into the analyser manifold for further reaction and/or measurement. The sample-to-extractant ratio can be varied within the limits normally applying to such operations, but the maximum concentration factor consistent with good operation is normally about 3 1. 

The period that elapses before two or more solutions are thoroughly mixed in a chemical kinetic experiment. In most manually controlled chemical kinetic studies, the mixing time is rarely a factor affecting accurate data acquisition however, the mixing time can be significant in rapid kinetic processes studied by continuous and stopped-flow kinetic techniques. 

Peracetic Acm-AcErALDEHYDE Reaction. The cobalt- and manganese-catalyzed reactions of peracetic acid with acetaldehyde were studied by a continuous flow technique (9). Peracetic acid (0.15M in acetic acid) and acetaldehyde-catalyst solutions were metered through rotameters to a mixing T (standard 0.25-inch stainless steel Swagelok T) and... 

There are, however, a number of disadvantages to using continuous flow techniques to study the kinetics of reactions on soil constituents. Often the colloidal particles are not dispersed—for example, the time required for an adsorptive to travel through a thin layer of collodial particles is not equivalent at all locations of the layer. Consequently, mass transfer can be significant if the sample is not dispersed. Skopp and McAllister (1986) note that even if the sample is dispersed, different pore and particle sizes of the adsorbent may result in nonuniform tracer transit times. The thickness of the disc of colloidal particles should be thin and measured to establish that perfect mixing is operational. 

The stirred-flow technique is an improvement over the continuous flow method described earlier. The method effects perfect mixing (Seyfried et al, 1988) so that the chamber and effluent concentrations are euqal and transport phenomena are minimized significantly. Additionally, the sorbent is dispersed and the dilution error present in the continuous-flow method can be accounted for. The stirred-flow technique also retains the attractive features of removing desorbed species at each step of the reaction process and of easily studying desorption kinetics phenomena. 

Reaction of NO with Sulfite and Bisulfite. We have recently studied the reactions of NO + S03Z and NO + HS03 using rapid-mixing continuous-flow and stopped-flow techniques in conjunction with UV spectrophotometry for detection of the reaction s product, N-nitrosohy-droxylamine-N-sulfonate (NHAS). 

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