Reaction-based chemical analysis in capillary electrophoretic systems by electrophoretically mediated microanalysis
Abstract
The performance of on-line homogeneous reaction-based chemical analysis in capillary electrophoretic systems by the methodology of electrophoretically mediated microanalysis (EMMA) is described. In EMMA, electrophoretic mixing is utilized to merge zones containing the analyte and its analytical reagents without the concurrent dilution and turbulence inherent in additive mixing methods; the reaction is then allowed to proceed either in the presence or the absence of an applied potential; and, finally, the reaction product is transported under the influence of an applied electric field to the detector. Detailed mathematical models are presented which describe the EMMA technique in terms of the four required steps for a reaction-based chemical analysis: (1) analyte and analytical reagent metering, (2) initiation of the reaction, (3) control of the reaction conditions and product formation, and (4) detection of the product. The theory is confirmed by experimental kinetic determinations of alcohol dehydrogenase. If an analyte and its product differ in electrophoretic mobility, the detection time of an EMMA product profile can be selectively manipulated by controlling when the analytical reaction is allowed to occur. This unique selectivity of the EMMA technique is described for the resolution of product profiles from non-reacting matrix interferants and for the simultaneous determinations of enzymes and substrates. The analytical merit of the EMMA method is evaluated for the endpoint determination of ethanol. The technique typically offers relative standard deviations of about 4% and 2% for determinations without and with an internal standard, respectively. Calibrations curves yield correlation coefficients of approximately 0.997 and linear ranges which extend to that quantity of substrate which can be reacted prior to passing by the detection position. Furthermore, the EMMA technique yields indistinguishable values from the standard spectrophotometric method. Moving boundary EMMA is presented as an alternative sample introduction technique. This method offers an order of magnitude greater sensitivity than zonal injection procedures, and its ease of implementation facilitates rapid analysis as the use of elevated electric field strengths and short capillaries yields a 24 s kinetic determination of leucine aminopeptidase.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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