Date of Thesis

2015

Description

Creatinine is a byproduct of muscle contraction in the body. It enters the bloodstream and then is filtered by the kidney. The Jaffé reaction is used clinically to measure creatinine levels in biofluids and is used as a measure of renal function. The neutral colorless creatinine molecule (0.05-20 mM) reacts with yellow anionic picrate (47 mM) to form the red Janovsky complex, also called the Jaffé product. Here, the Jaffé reaction is carried out in-capillary using electrophoretically mediated microanalysis (EMMA) and transient isotachophoretic (tITP) stacking. In addition, computer simulations of the EMMA/tITP dynamics enable optimization of these analyses. An aqueous, low conductivity matrix for creatinine-containing samples is shown to result in a smaller Jaffé product peak than when creatinine in a more conductive matrix. Also, to improve reproducibility between experiments with tITP stacking (challenged by changes in electroosmotic flow [EOF] affecting the timing of the reaction) this work has turned to computer simulations to provide insight into the ionic migration dynamics of the Jaffé reaction system. Simulations performed with computer program SIMUL 5.0 allow for the dynamics of the Jaffé reaction to be observed along the entire length of the simulated capillary to visualize the Jaffé product peak size and shape over the course of the analysis with hydroxide-mediated tITP stacking. SIMUL has shown that the optimal detection time and Jaffé product height is affected by the length and concentration of the hydroxide plug. Also, due to disagreement about Jaffé product structure in the literature, mass spectrometry provides insight into the structure of the Jaffé product, suggesting a 1:1 complex.

Keywords

Jaffé reaction, Capillary electrophoresis (CE), Electrophoretically mediated microanalysis (EMMA), Transient isotachophoretic (tITP) stacking, SIMUL, Mass spectrometry (MS)

Access Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science

Major

Cell Biology/Biochemistry

First Advisor

Timothy G. Strein

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