Velocity, ionic experimental determination

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

One can expect (see fine print further) that the greater diffusivity of the counter-ion layer as compared to that established in Helmholtz model, would only affect the velocity distribution profile of the displacement of individual fluid layers in the direct vicinity of the solid surface. The experimentally observed velocity of the mutual motion of the phases with respect to each other, v0, determined, as in Helmholtz model, by the potential change significantly (curve 2 approaches the same limiting value as curve 7 ). This is also confirmed by the fact that the distance between the capacitor plates, 8, which is the only parameter defining the geometry of the system in the Helmholtz model, is not present in the final expression.4 The thickness of the ionic atmosphere, k 1, may be used as the parameter closest to the distance 8, i.e. 8=1/k. 

Recently a method has been developed to determine the average flow velocity by measuring the electric current in a microchannel under electroosmotic flow [6, 7]. The experimental setup is shown in Fig. 2 and consists of a microchannel, a high-voltage power supply, and a data acquisition system. The microchannel and reservoir 2 are initially filled with an electrolyte solution that has a slightly different concentration than reservoir 1 (cj — C2 = 5 %). The concentration difference must be small so that the zeta potential and ionic concentrations are nearly uniform throughout the microchannel. 

In theory, there are no limits to the accessibility of electrokinetic data. However, there are a number of physical situations which limit the range of electrokinetic data which can be obtained in the above described experimental set-ups. The experimental techniques described above require visual determination of particle velocities and are typically limited to the range 3-100 pm/s. Additionally, according to equation (19.24), current and solution conductivity affect the particle mobility. In experimental practice, the limit for current in the cell is around 300 pA with the use of blank platinum electrodes. Under higher currents, electrolytic reactions at the electrodes result in electrode polarization, heating and subsequent formation of gas bubbles and thermal convection in the cell. Furthermore, solution conductivity measurements are not reliable below about 10 pS/cm. The above limitations restrict the typical range of solution ionic strengths at which one can work to O.l-lOOmM. 

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