Setting Up the Reaction Conditions

Some additional modifications of the reaction conditions used with other assay methods may be required to proceed with an HPLC assay. One change is related to the total volume of the reaction mixture. Because the HPLC assay is basically a discontinuous technique, obtaining kinetic data requires multiple samples, each one representing a single time point. Traditionally, reactions requiring multiple sampling have been arranged in one of two ways. In one arrangement (Fig. 4.10/1), separate reaction mixtures are set up, each one representing a single time point. In this case, the total volume required for a single reaction mixture is the volume required for a single injection. The number of incubation tubes would be determined by the number of time points required by the experiment. In the second arrangement (Fig. 4.105), a single incubation mixture is prepared, and samples are removed from it at suitable intervals for analysis. In this arrangement, the volume required for the reaction mixture would be determined as the product of the volume needed for each injection multiplied by the total number of injections.

Since with both arrangements the volume of a single injection is the important variable, it would appear that once this value has been determined, the overall reaction volume can be established. However, another variable, the type of injector to be used, must also be considered.

Injectors are of two basic designs. Those of the type illustrated in Figure 4.11A require that a sample of known volume be removed from the reaction

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