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.

Significance of the Deformation History within the Hinge Zone of the Pennsylvania Salient, Appalachian Mountains

Two competing models exist for the formation of the Pennsylvania salient, a widely studied area of pronounced curvature in the Appalachian mountain belt. The viability of these models can be tested by compiling and analyzing the patterns of structures within the general hinge zone of the Pennsylvania salient. One end-member model suggests a NW-directed maximum shortening direction and no rotation through time in the culmination. An alternative model requires a two-phase development of the culmination involving NNW-directed maximum shortening overprinted by WNW-directed maximum shortening. Structural analysis at 22 locations throughout the Valley and Ridge and southern Appalachian Plateau Provinces of Pennsylvania are used to constrain orientations of the maximum shortening direction and establish whether these orientations have rotated during progressive deformation in the Pennsylvania salient's hinge. Outcrops of Paleozoic sedimentary rocks contain several orders of folds, conjugate faults, steeply dipping strike-slip faults, joints, conjugate en echelon gash vein arrays, spaced cleavage, and grain-scale finite strain indicators. This suite of structures records a complex deformation history similar to the Bear Valley sequence of progressive deformation. The available structural data from the Juniata culmination do not show a consistent temporal rotation of shortening directions and generally indicate uniform,