Maximizing Product Formation and Minimizing Degradation: A Look at Buffering Conditions and Post-Reaction Degradation for the In-Line Jaffe Reaction with Capillary Electrophoresis

Creatinine levels in blood serum are typically used to assess renal function. Clinical determination of creatinine is often based on the Jaffe reaction, in which creatinine in the serum reacts with sodium picrate, resulting in a spectrophotometrically quantifiable product. Previous work from our lab has introduced an electrophoretically mediated initiation of this reaction, in which nanoliter plugs of individual reagent solutions can be added to the capillary and then mixed and reacted. Following electrophoretic separation of the product from excess reactant(s), the product can be directly determined on column. This work aims to gain a detailed understanding of the in-capillary reagent mixing dynamics, in-line reaction yield, and product degradation during electrophoresis, with an overall goal of improving assay sensitivity. One set of experiments focuses on maximizing product formation through manipulation of various conditions such as pH, voltage applied, and timing of the applied voltage, in addition to manipulations in the identity, concentration, and pH of the background electrolyte. Through this work, it was determined that dramatic changes in local voltage fields within the various reagent zones lead to ineffective reagent overlapping. Use of the software simulation program Simul 5 enabled visualization of the reaction dynamics within the capillary, specifically the wide variance between the electric field intensities within the creatinine and picrate zones. Because of this simulation work, the experimental method was modified to increase the ionic strength of the creatinine reagent zone to lower the local voltage field, thus producing more predictable and effective overlap conditions for the reagents and allowing the formation of more Jaffe product. As second set of experiments focuses on controlling the post-reaction product degradation. In that vein, we have systematically explored the importance of the identity, concentration, and pH of the background electrolyte on the post-reaction degradation rate of the product. Although prior work with borate background electrolytes indicated that product degradation was probably a function of the ionic strength of the background electrolyte, this work with a glycine background electrolyte demonstrates that degradation is in fact not a function of ionic strength of the background electrolyte. As the concentration and pH of the glycine background increased, the rate of degradation of product did not change dramatically, whereas in borate-buffered systems, the rate of Jaffe product degradation increased linearly with background electrolyte concentration above 100.0 mM borate. Similarly, increasing pH of the glycine background electrolyte did not result in a corresponding increase in product degradation, as it had with the borate background electrolyte. Other general trends that were observed include: increasing background electrolyte concentration increases peak efficiency and higher pH favors product formation; thus, it appears that use of a background electrolyte other than borate, such as glycine, the rate of degradation of the Jaffe product can be slowed, increasing the sensitivity of this in-line assay.

Understanding Electrophoretic Stacking Of In-Capillary Generated Reaction Products

The separation of small molecules by capillary electrophoresis is governed by a complex interplay among several physical effects. Until recently, a systematic understanding of how the influence of all of these effects is observed experimentally has remained unclear. The work presented in this thesis involves the use of transient isotachophoretic stacking (tITP) and computer simulation to improve and better understand an in-capillary chemical assay for creatinine. This assay involves the use of electrophoretically mediated micro-analysis (EMMA) to carry out the Jaffé reaction inside a capillary tube. The primary contribution of this work is the elucidation of the role of the length and concentration of the hydroxide plug used to achieve tITP stacking of the product formed by the in-capillary EMMA/Jaffé method. Computer simulation using SIMUL 5.0 predicts that a 3-4 fold gain in sensitivity can be recognized by timing the tITP stacking event such that the Jaffé product peak is at its maximum height as that peak is electrophoresing past the detection window. Overall, the length of the hydroxide plug alters the timing of the stacking event and lower concentration plugs of hydroxide lead to more rapidly occurring tITP stacking events. Also, the inclusion of intentional tITP stacking in the EMMA/Jaffé method improves the sensitivity of the assay, including creatinine concentrations within the normal biological range. Ultimately, improvement in assay sensitivity can be rationally designed by using the length and concentration of the hydroxide plug to engineer the timing of the tITP stacking event such that stacking occurs as the Jaffé product is passing the detection window.