Serum creatinine levels are an important and oft-measured indicator of renal function. The Jaffe reaction of creatinine with sodium picrate yields a colored product which is the basis for a routine clinical analysis. The electrophoretically mediated initiation of this reaction utilizing nanoliter volumes in a capillary column has been previously reported. This work involves a fundamental and quantitative investigation of the factors influencing the reaction yield and the transient isotachophoretic stacking of the Jaffe product. Factors studied include the effects of borate versus glycine buffer systems, the role and positioning of excess hydroxide and chloride leading electrolyte zones, and the timing of the applied voltage used to overlap the reactant zones. Addition of an initial discrete hydroxide zone is found to dramatically enhance the reaction yield as well as induce t-ITP stacking under certain conditions, while addition of chloride to the creatinine zone is found to yield a significant improvement in stacking behavior, presumably owing to minimized electrodispersion of the product. Novel insights from the use of simple pressure-driven flow to obtain a profile of the reaction zone will also be reported.