ABSTRACT
A large number of analytically and physiologically significant enzymes produce or consume hydrogen peroxide (H2O2). Post-column reaction of H2O2 with luminol in an alkaline solution results in chemiluminescence. A prototype reactor/detector for CE with chemiluminescent detection (CLD) of H2O2 was constructed and utilized. Two variations of the detection cell illustrated the difference between an analyte-limiting (stagnant-cell) and a window-limiting (swept-cell) detection scheme. The stagnant-cell detector had a low limit of detection (39 fmol at S/N = 3), but poor separation efficiency (1100 theoretical plates). The swept-cell detector demonstrated greater efficiency (226,000 theoretical plates), but a detection limit one order of magnitude higher (380 fmol). Several enzyme assays were developed using electrophoretically mediated microanalysis (EMMA) with chemiluminescent monitoring of H2O2. EMMA can be used to perform enzyme assays in a capillary format using electrophoretic mixing. Accumulated product of an incubation is swept to the detector and traditionally monitored by UV-VIS absorbance, fluorescence, or electrochemical detection. Higher sensitivity may also be obtained from the use of CLD. Chemical amplification with galactose oxidase and glucose oxidase resulted in the production of H2O2, while catalase enzymatically consumed H2O2. This work showed a CLD limit of 9300 molecules in the case of catalase.
Subject(s)
Catalase/analysis , Electrophoresis, Capillary/methods , Galactose Oxidase/analysis , Glucose Oxidase/analysis , Electrophoresis, Capillary/instrumentation , Hydrogen Peroxide , Indicators and Reagents , Luminescent Measurements , Luminol , Microchemistry/instrumentation , Microchemistry/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Ensemble averaging and digital filtering were implemented for signal-to-noise ratio improvement in the separation techniques of size-exclusion chromatography, immunoaffinity chromatography, capillary zone electrophoresis and capillary ion analysis. Results of ensemble averaging were always greater than statistically predicted. Techniques included five to nine replicate separations and yielded signal-to-noise improvement factors of 2.5 to 9.3. Running-average and time constant (RC)-convolution digital filters yielded increases in the signal-to-noise ratio ranging from zero to twelve. This paper will discuss and illustrate the usage of ensemble averaging and digital filtering in liquid-phase separation techniques.
