ABSTRACT
This study presents a high-performance capillary electrophoresis (CE) method to analyze five constituents of Puerariae radix, an important crude herb used in Chinese medicine. Puerarin, daidzin, daidzein, genistein and biochanin A are the bioactive constituents of Puerariae radix. Herein, those analytes were successfully separated within 6 min using a pH 10.1 borax-NaOH buffer. The effects of pH value and concentration of the running buffer on the separation of the five analytes were also examined. The relative standard deviations of the analytes' migration times were less than 0.38% under the optimized separation conditions. Notably, the correlation coefficients of the analytes' linear calibration graphs exceeded 0.998. Moreover, the amounts of the five constituents in three different Puerariae radix samples were determined by the CE method with a relatively simple extraction procedure.
Subject(s)
Drugs, Chinese Herbal/chemistry , Electrophoresis, Capillary/methods , Isoflavones/analysis , Plant Roots/chemistry , Genistein/analysis , Genistein/chemistry , Hydrogen-Ion Concentration , Isoflavones/chemistry , Osmolar ConcentrationABSTRACT
Restriction mapping is one of the essential steps in gene analysis and molecular biology studies. Slab gel electrophoresis is the traditional way to separate DNA fragments for restriction mapping. However, slab gel electrophoresis does not provide sufficient resolution as required in many mapping applications, and the use of radioisotopes in traditional mapping methods creates health hazards. In the present study, capillary electrophoresis coupled with laser-induced fluorescence detection and a modified partial digestion mapping procedure was developed to map DNA fragments. By using capillary electrophoresis, a restriction map of genomic lambda phage clone of human interleukin 5 receptor alpha chain (IL5R alpha) gene was constructed. The IL5R alpha gene was analyzed to have five XbaI enzyme cutting sites at locations 1370, 2290, 2950, 5430, and 9330. The system was further characterized by using pBluescript SK(+) phagemid DNA as a model. Using a sequence-derived map as a reference, the pBluescript SK(+) restriction map constructed by capillary electrophoresis had an accuracy greater than 90%.
Subject(s)
DNA/chemistry , Electrophoresis, Capillary/methods , Restriction Mapping , Humans , Lasers , Receptors, Interleukin/genetics , Receptors, Interleukin-5 , Spectrometry, FluorescenceABSTRACT
This paper presents a rapid and reliable micellar electrokinetic capillary chromatography (MEKC) method to separate major prostaglandins and thromboxane B2. The running buffer was modified with sodium dodecyl sulfate (SDS). The effects of the SDS concentration on the migration behavior of analytes was also examined. Moreover, the influences of electrolyte concentration and capillary temperature on the separation were investigated. In optimum conditions, seven major prostaglandins and thromboxane B2 could be separated within 8 min. The relative standard deviations of the migration times (reproducibility) of the analytes were less than 0.82%.
Subject(s)
Electrophoresis, Capillary/methods , Prostaglandins/analysis , Electrolytes/chemistry , Methanol/chemistry , Micelles , Osmolar Concentration , Prostaglandins/chemistry , Reference Standards , Reproducibility of Results , Sodium Dodecyl Sulfate/chemistry , Spectrophotometry, Ultraviolet , Surface-Active Agents/chemistry , Temperature , Thromboxane B2/analysis , Thromboxane B2/chemistry , Time FactorsABSTRACT
The band dispersion phenomena in capillary zone electrophoresis (CZE) using untreated and surface-treated open tubular and gel-filled capillaries were experimentally evaluated, with emphasis on small capillary diameters (10-100 microns). Laser-induced fluorescence detection was used for high-sensitivity detection of the isoindoles originated from model amino acids. The plots of plate height vs electric field strength were generated for different column radii and compared with a theoretical model for CZE. In addition to the diffusion-controlled band dispersion in the relatively low electric field range, adsorptive interactions between a solute and the capillary wall may play a certain role in band-broadening. The sorption-desorption kinetics become important with increasing electric field strength. Thermal effects appear to contribute little to band-broadening in relatively small capillaries (less than 50-microns i.d.) within normal operating voltages (less than 30 kV), but could become significant in capillaries with larger bores (greater than 75-microns i.d.). With gel-filled capillaries of small diameters (less than 50-microns i.d.), diffusion processes can be minimized. In addition, thermal effects do not appear critical in such columns at reasonable voltages.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Animals , Electrophoresis, Polyacrylamide Gel/instrumentation , HumansABSTRACT
Amino acids and peptides, from both standard solutions and biological samples, were successfully reacted with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde, at low concentration, to form highly fluorescent isoindole derivatives. The formed mixtures are effectively separated by high-performance capillary electrophoresis and their constituents detected by their laser-induced fluorescence signals. The minimum detectable quantities in the low attomole (10(-18) mol) range are encountered.
Subject(s)
Amines/analysis , Benzoates/chemistry , Quinolines/chemistry , Amino Acids/analysis , Electrophoresis , Hydrogen-Ion Concentration , Indicators and Reagents , Lasers , Peptides/analysis , Spectrometry, FluorescenceABSTRACT
The potential clinical diagnostic utility of capillary electrophoresis in an open-tubular column is established. Separation patterns for serum proteins by conventional agarose gel electrophoresis can be faithfully reproduced by a capillary electrophoresis procedure that provides the complete run data in 8 min. Hemoglobin variants can be separated within 10 min. The capillary electrophoresis separations are performed reliably and reproducibly in an untreated 75 micron (i.d.) x 25 cm fused-silica column. Diluted serum or hemoglobin samples can be loaded on an automated instrument with on-line injection, detection, and quantification, providing a truly "walkaway" electrophoresis system.
Subject(s)
Electrophoresis/methods , Blood Proteins/isolation & purification , Capillary Action , Cerebrospinal Fluid Proteins/isolation & purification , Electrophoresis, Agar Gel , Hemoglobins, Abnormal/isolation & purification , Humans , Proteinuria/urineABSTRACT
3-(2-Furoyl)quinoline-2-carbaldehyde (FQCA) has been synthesized and characterized as a fluorogenic derivatizing reagent for liquid chromatography. The reagent forms highly fluorescent isoindoles upon reaction with primary amines. The spectral properties and optimum reaction conditions for the formation of isoindoles have been investigated. The utility of FQCA for the high-sensitivity analysis of amino acids by laser-induced fluorescence with an argon-ion laser is demonstrated.
Subject(s)
Amines/analysis , Chromatography, Liquid/methods , Furans , Quinolines , Fluorescence , LasersABSTRACT
3-Benzoyl-2-quinolinecarboxaldehyde has been synthesized and characterized for use as a precolumn fluorogenic reagent for the ultrahigh sensitivity determination of primary amines by micro-column liquid chromatography with laser-induced fluorescence detection. The reaction conditions and the spectral properties of the derivatives were investigated with standard amino-acids. The detection limits, with an HeCd laser operated at 442 nm, are in the low femtogram range. The linear dynamic range is at least three orders of magnitude. The separation of a standard amino-acid mixture and the high-sensitivity analysis of a hydrolysed protein sample are demonstrated.
