A Novel Enzyme-immobilized Detection Interface For Capillary Electrophoresis-chemiluminescence Analysis And Its Application In Pharmaceutical Analysis

With the rapid development of life science and clinical pharmacy, pharmaceutical analysis plays a more and more important role in analytical science. The analysis of trace amounts of biomolecules and drugs in complex biological matrixes is a major problem troubles analyst. Although capillary electrophoresis (CE) has existed for decades, this efficient separation technology still remains highly active for research due to its high separation efficiency, short run time and avoidance of toxic organic solvent. The different modes of operation in CE have provided the analyst with many options for handling a wide range of solute types on a single piece of equipment. However, due to the ultra-small sampling volume for these CE methodologies, a highly sensitive detector is required to obtain acceptable detection ability. Although mass spectrometry and laser-induced fluorescence provide satisfying sensitivity, their extensive applications are limited by the high expense of the instrumentation and the great complexity of the assay procedure. Alternatively, using an enzyme reaction with a unique high catalyzing efficiency to greatly amplify the detection signal has attracted increasing interest in CE. Enzyme-catalyzed chemiluminescence (CL) detection, with advantage of simplicity and high sensitivity, has been considered to be ideally suitable for CE.Given the above facts, we designed a novel enzyme-immobilized flow-through interface for CE-CL detection. Its main contents are introduced as followings:Part1Design a novel enzyme-immobilized flow cell used as end-column CL detection interface in CE.A novel enzyme-immobilized flow-through interface was designed for sensitive end-column CL detection in CE. Enzyme was covalently bound on an aldehyde-actived polymer membrane immobilized in a flow cell to catalyze the CL reaction that occurred in it. Luminol was effluented from the CE column and triggered a horseradish peroxidase-catalyzed CL reaction, to produce an enhanced detection signal. Luminol as the the model analyte can be assayed in the range of0.1to300μM (R2=0.9913). The RSDs for the migration time, the peak heights were less than7.5%and8.2%. After50successive assays, the CL signal also retained acceptable repeatability and stabilityPart2thiolated β-cyclodextrin-modified gold nanoparticles (CD-GNPs) coated capillary for open-tublular capillary electrochromatography (OTCEC)-CL detection of biogenic amines.Thiolated β-cyclodextrin as active group, gold nanoparticle as a support, CD-GNPs was synthesized. CD-GNPs as a stationary phase were coated on the inner surface of capillary to prepare OTCEC column by alkanethiol self-assembly process. This OTCEC was adopted to improve the separation efficiency. The enzyme-immobilized detection interface and luminol-hydrogen peroxide CL system continued to use in this work. Using glycine as the model analyte, N-(4-Aminnobutyl)-N-ethylisoluminol-derivatized glycine was effluented from the OTCEC column and triggered CL reaction. Glycine can be assayed in the range of0.5to200μM (R2=0.9921) with a detection limit of0.12μM (S/N of3). The whole analysis process can be completed within13min with a theoretical plate number of22500. Human saliva and urine samples were also measured by this method.