A novel enzyme-immobilized flow-through interface was designed for sensitive end-column chemiluminescent (CL) detection in open-tubular capillary electrochromatography (OTCEC). Enzyme was covalently bound on an aldehyde-activated polymer membrane immobilized in a flow cell to catalyze the CL reaction that occurred in it. Using glycine as the model analyte, N-(4-aminobutyl)-N-ethylisoluminol-derivatized glycine effused from the OTCEC column and triggered a horseradish peroxidase (HRP)-catalyzed CL reaction, to produce an enhanced detection signal. To obtain a satisfying result for complex biological sample analysis, a thiolated β-cyclodextrin-modified gold nanoparticles-coated OTCEC column was adopted to improve the separation efficiency. Glycine can be assayed in the range of 0.50-200 μM (R(2) = 0.9921) with a detection limit of 0.12 μM (S/N of 3). The whole analysis process can be completed within 13 min with a theoretical plate number of 22,500. Compared to the previously reported solution-phase enzyme catalysis, pre-column and on-column immobilized enzyme catalysis for capillary electrophoresis detection, a significantly reduced enzyme consumption and greatly improved enzyme stability can be achieved with the use of this end-column enzyme-immobilized detection interface. The novel flow cell can be further applied in other capillary electrophoresis modes including capillary zone electrophoresis, capillary gel electrophoresis, and micellar electrokinetic chromatography. It is also suitable for some other detectors such as fluorimetric, ultraviolet-visible absorption spectrometric and electrochemical detectors.
A transition metal chelate unstable at a high oxidation state, diperiodatocuprate (III) (K₅[Cu(HIO₆)₂], DPC), was synthesized and applied in the luminol-based chemiluminescence (CL) system for highly sensitive CE end-column detection of dopamine (DA). This method was based on the fact that DA enhanced the CL emission resulting from the reaction between luminol and DPC in alkaline medium. The DPC-luminol-DA CL system showed very intensive emission and very fast kinetic characteristics, thus resulting in a high sensitivity in flow-through detection mode for CE. Under optimal conditions, the linear range was 1.0 × 10⁻⁸-5.0 × 10⁻⁵ g/mL (R² = 0.9984) with a limit of detection of 6.0 × 10⁻⁹ g/mL (S/N = 3). The RSDs of the peak height and the migration time were about 4.2 and 2.4% for a standard sample at 3.0 × 10⁻⁶ g/mL (n = 5), respectively. The presented method has been successfully used for the determination of DA in commercial preparation and human urine samples after clean-up using SPE.
Luminol-type compounds can be used as chemiluminescent (CL) derivatization reagents for amines, carboxylic acids and protein. Copper chelate diperiodatocuprate(III) (K5[Cu(HIO6)2], DPC) was synthesized by complexation of copper at trivalent oxidation state and periodate in a strong basic medium. It was found that DPC can greatly enhance the reaction between luminol-type compounds and H2O2 to produce very strong CL emission. Based on this fact, a rapid CE method combined with high-sensitive end-column CL detection was established to simultaneously analyze luminol and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) with wide concentration range of 3.0-300 nmol/L in 5 min. The RSDs of the signal intensity and the migration time were less than 3.9 and 7.0% for a standard sample containing 100 nmol/L luminol and ABEI (n=5), respectively. The investigation implies that DPC is a promising sensitizer for CE-CL detection of a great variety of biomolecules and drugs in biological samples after derivatization using luminol derivatives.
A simple, fast and sensitive capillary electrophoresis (CE) strategy combined with chemiluminescence (CL) detection for analysis of ofloxacin (OF) enantiomers was established in the present work. Sulfonated β-cyclodextrin (β-CD) was used as the chiral additive being added into the running buffer of luminol–diperiodatocuprate (III) (K5[Cu(HIO6)2], DPC) chemiluminescence system. Under the optimum conditions, the proposed method was successfully applied to separation and analysis of OF enantiomers with the detection limits (S/N=3) of 8.0 nM and 7.0 nM for levofloxacin and dextrofloxacin, respectively. The linear ranges were both 0.010–100 μM. The method was utilized for analyzing OF in urine; the results obtained were satisfactory and recoveries were 89.5–110.8%, which demonstrated the reliability of this method. This approach can also be further extended to analyze different commercial OF medicines.
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