ABSTRACT
A laboratory made capillary electrophoresis (CE) system using shorter capillary column was designed and constructed in our research group. This promising CE system was then connected with confocal fluorescence microscopy which was also developed in our research group. In order to show the applicability of this new system on chiral separation of DL-tryptophan (DL-Trp), studies were undertaken using cyclodextrins (CDs) and their derivatives; sulfated (S-CDs) and highly sulfated cyclodextrins (HS-CDs) as chiral selectors. Different selector concentrations were tested at various pH levels (pH 2.5, 4.0, 6.0, 9.0) to optimize the separation conditions. The best results were achieved at lower pH values (e.g. pH 2.5 and pH 4.0). HS-beta-CD and HS-gamma-CD were found to be the most effective chiral selectors with a shorter retention time at low selector concentrations.
Subject(s)
Electrophoresis, Capillary/methods , Tryptophan/chemistry , Cyclodextrins/chemistry , Hydrogen-Ion Concentration , StereoisomerismABSTRACT
The design and construction of a miniaturized CE system has been developed for the analysis of clinically important compounds; serotonin and rac-propranolol. Chiral separation of rac-propranolol was investigated by using native and derivatized CDs at varios pH levels (e.g. 2.5, 4.0, 6.0, 9.0). Confocal fluorescence microscopy was used for the native direct detection of target compounds. Analysis time obtained under the optimum separation conditions was 84 s. LOD and LOQ values of serotonin were found as low as 22 and 72.6 fg/microL while these limits were 44 and 145.2 fg/microL for popranolol enantiomers, respectively. LOD and LOQ values were found to be the lowest limits reported before. The calibration curves were linear in the concentration range of 40-1500 fg/microL with the determination coefficients between 0.9994 (+/-0.01) and 0.9997 (+/-0.02).
Subject(s)
Electrophoresis, Capillary/methods , Propranolol/analysis , Serotonin/analysis , Cyclodextrins/chemistry , Fluorescence , Hydrogen-Ion Concentration , Propranolol/isolation & purification , Reproducibility of Results , Serotonin/isolation & purification , Stereoisomerism , Ultraviolet RaysABSTRACT
Lysozyme (14.4 kDa), BSA (66.4 kDa) and beta-galactosidase (116 kDa) were successfully separated and determined directly within 80 seconds using a laboratory-made miniaturized capillary electrophoresis apparatus provided with a confocal fluorescence spectrometry. Several parameters controlling on the detection limits, including focusing effect, laser power and buffer composition were tested and optimized. Separation buffer was 10 mM phosphate containing 4 mM CTAB at pH 2.5. The LOD values for lysozyme, beta-galactosidase and BSA were found as 9.0, 13.0 and 55 fg/microl, respectively. This miniaturized CE system offers a lot of advantages for protein analysis. First; it provides reproducible separation reducing wall-adsorption effects at low pH. Second, the analysis time observed from our system is in the range of chip electrophoresis applications. And finally, LOD values for standard proteins are much more lower than that of obtained from traditional gel electrophoresis method.
Subject(s)
Electrophoresis, Microchip/instrumentation , Electrophoresis, Microchip/methods , Muramidase/analysis , Serum Albumin, Bovine/analysis , beta-Galactosidase/analysis , Animals , Cattle , Chickens , Escherichia coli/enzymology , Fluorescence , Microscopy, Confocal , Ovum/enzymology , Sensitivity and SpecificityABSTRACT
The present study reports the investigation of capillary electrophoresis (CE) for the separation of the photosynthetic pigments (chlorophyll derivatives as well as carotenoids) together. Various CE methods, such as micellar electrokinetic chromatography, capillary electrokinetic chromatography, and nonaqueous capillary electrophoresis (NACE) are tested, with coated and uncoated capillary columns to evaluate optimal separation conditions using diode array detection. The effect of different type and composition of organic solvents and surfactants on the separation is discussed. Detection limits are found in the range of 1.14-2.45 ppm. According to the system suitability results, the most effective separation is observed using NACE with Aliquat 336 as cationic surfactant in coated capillary and mixture of MeOH-ACN-THF (5:4:1, v/v/v) as solvent. Quantitative evolution is investigated, and recovery percentage values are found to be 96.7-102%.
Subject(s)
Carotenoids/isolation & purification , Electrophoresis, Capillary/methods , Porphyrins/isolation & purification , Chromatography, Micellar Electrokinetic Capillary/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Capacitively coupled contactless conductivity detection (C(4)D) is a new technique providing high sensitivity in capillary electrophoresis (CE) especially for small ions that can otherwise only be determined with indirect methods. In this work, direct determination and validation of valproic acid (VPA) in biological fluids was achieved using CE with C(4)D. VPA is of pharmacological interest because of its use in epilepsy and bipolar disorder. The running electrolyte solution used consisted of 10mM 2-(N-morpholino)ethane sulfonic acid (MES)/dl-histidine (His) and 50microM hexadecyltrimethylammonium bromide (HTAB) at pH 6.0. Caproic acid (CA) was selected as internal standard (IS). Analyses of VPA in serum, plasma and urine samples were performed in less than 3min. The interference of the sample matrix was reduced by deproteinization of the sample with acetonitrile (ACN). The effect of the solvent type and ratio on interference was investigated. The limits of detection (LOD) and quantitation (LOQ) of VPA in plasma samples were determined as 24 and 80ng/ml, respectively. The method is linear between the 2 and 150microg/ml, covering well the therapeutic range of VPA (50-100microg/ml).
