The application of spline wavelet transform to the determination of electroosmotic mobility in capillary electrophoresis

The rule of current change was studied during capillary electrophoresis (CE) separation process while the conductivity of the sample solution was different from that of the buffer. Using a quadratic spline wavelet of compact support, the wavelet transforms (WTs) of capillary electrophoretic currents were performed. The time corresponding to the maximum of WT coefficients was chosen as the time of current inflection to calculate electroosmotic mobility. The proposed method was suitable for different CE modes, including capillary zone electrophoresis, nonaqueous CE and micellar electrokinctic chromatography. Compared with the neutral marker method, the relative errors of the developed method for the determination of electroosmotic mobility were all below 2.5%. (C) 2002 Elsevier Science B.V. All rights reserved.

Modeling and optimization for separation of ionic solutes in pressurized flow capillary electrochromatography

By manipulation of applied pressure or voltage, pressurized flow capillary electrochromatography (P-CEC) permits unique control of selectivity for ionic solutes. A simple mathematical model has been developed to describe the quantitative relationship between the electrochromatographic retention factor (k(*)) of charged solutes and the applied voltage and pressure. The validity of the model was verified experimentally with hydrophilic interaction mode CEC (HI-CEC). On the basis of the model developed, it was found that the value of k(*) could be predicted accurately using only a limited number of data points from the initial experiments at different voltages or pressures. Correlation between the experimentally measured and calculated k(*) was excellent, with a correlation coefficient greater than 0.999. Optimization for the separation of peptides by P-CEC was also performed successfully on the basis of the proposed model.

Separation of enantiomers by nanoliquid chromatography and capillary electrochromatography using a bonded cellulose trisphenylcarbamate stationary phase

A cellulose trisphenylcarbamate-bonded chiral stationary phase was applied to nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) with nonaqueous and aqueous solutions as the mobile phases. Several chiral compounds were successfully resolved on the prepared phase by nano-LC. The applicability of nonaqueous CEC on a cellulose derivative stationary phase was investigated with the organic solvents methanol, hexane, 2-propanol, and tetrahydrofuran (THF) containing acetic acid, as well as triethylamine as the mobile phases. Enantiomers of warfarin and praziquantel were baseline-resolved with plate numbers of 82 300 and 38 800 plates/m, respectively, for the first eluting enantiomer. The influence of applied voltage, concentration of nonpolar solvent, apparent pH, and buffer concentration in the mobile phase on the electroosmotic flow (EOF) and the mobility of the enantiomers was evaluated. Enantioseparations of traps-stilbene oxide and praziquantel were also achieved in aqueous CEC with plate numbers of 111 100 and 107 400 plates/m, respectively, for the first eluting enantiomer. A comparison between nonaqueous CEC and aqueous CEC based on a cellulose trisphenylcarbamate stationary phase was discussed. Pressure-assisted CEC was examined for the chiral separation of praziquantel and faster analysis with high enantioselectivity was acquired with the proper pressurization of the inlet vial.

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column

The mixed mode of reversed phase (RP) and strong canon-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for to and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.

Study of competitive binding of enantiomers to protein by affinity capillary electrochromatography

Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule. (C) 2002 Elsevier Science B.V. All rights reserved.

Characteristics of electroosmotic flow and migration of neutral solutes under stepwise gradient elution of capillary electrochromatography

A theoretical study on the velocity of electroosmotic flow (EOF) and the retention times of neutral solutes under multiple-step gradient of capillary electrochromatography (CEC) was carried out, focusing on that with three kinds of mobile phases. Through the model computations, the detaining time of the second kind of mobile phase in the column was proved to play an important role in affecting EOF. The variation speed of EOF was shown to be determined by the differences among dead times in different steps. In addition, the prediction of the retention times of 13 aromatic compounds under gradient mode was performed with the deduced equations. A relative error below 3.3% between the calculated and experimental values was obtained, which demonstrated the rationality of the theoretical deduction. Our study could not only improve the comprehension of stepwise gradient elution, but also be of significance for the further optimization of separation conditions in the analysis of complex samples.

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site. proved to be the preferential one, being 6.50 x 10(5) 1.94 x 10(6) and 8.94 x 10(5). respectively. In addition. it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions. (C) 2002 Elsevier Science B.V. All rights reserved.

DNA diagnosis by capillary electrophoresis and microfabricated electrophoretic devices

DNA diagnosis is experiencing an impressive progression towards the development of novel technology to identity various clinically relevant categories of genetic changes and to meet the exponential growth of genomics. The introduction of capillary electrophoresis has dramatically accelerated the completion of the first draft of the human DNA sequence in the Human Genome Project, and thus, has become the method of choice for analysis of various genetic variants. The recent development of microfabricated electrophoretic devices has led to the possibility of integrating multiple sample handling with the actual measurement steps required for automation of molecular diagnostics. This review highlights the most recent progress in capillary electrophoresis and electrophoretic microdevices for DNA-based diagnostics, including the important areas of genotyping for point mutation, single nucleotide polymorphisms, short tandem repeats and organism identification. The application of these techniques for infectious and genetic disease diagnosis, as well as forensic identification purpose, are covered. The promising development and the challenges for techinical problems are also discussed.

Evaluation of the interaction between netropsin and double stranded DNA by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to study the interaction between netropsin and a 14mer double stranded DNA (dsDNA). The binding constant of this interaction calculated from Scatchard plot was (1.07+/-0.10) X 10(5) (mol/L)(-1). The binding stoichiometry was 1:1. The use of polyacrylamide coated capillary showed better effect in the analysis of DNA than noncoated capillary.

Capillary electrochromatographic separation of enantiomers on chemically bonded type of cellulose derivative chiral stationary phases with a positively charged spacer

Positively charged chiral stationary phases (CSPs) were prepared for capillary electrochromatography (CEC) separation of enantiomers by chemically immobilizing cellulose derivatives onto diethylenetriaminopropylated silica (DEAPS) with tolylene-2,4-diisocyanate (TDI) as a spacer reagent. Anodic electroosmotic mobility was observed in both nonaqueous and aqueous mobile phases due to the positively charged amines on the surface of the prepared CSPs. For comparison, the traditionally used 3-aminopropyl silica (APS) was also adopted as the base material instead of DEAPS to prepare CSP. It was observed that the EOF on the DEAPS-based CSP was 18%-60% higher than that on the APS-based CSP under nonaqueous mobile phase conditions. Separation of enantiomers in CEC was performed on the positively charged CSPs with the nonaqueous mobile phases of pure ethanol or mixture of hexane-alcohol and the aqueous phases of acetonitrile-water or 95% ethanol. Fast separation of enantiomers was achieved on the newly prepared CSPs.

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregrarn is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.

Abnormal phenomenon of the dependence of the retention factors for uncharged species on applied voltage in capillary electrochromatography

Polymethacrylate-based monolithic columns were prepared for capillary electrochromatography (CEC) by in situ copolymerization of butyl methacrylate (BMA), 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and ethylene dimethacrylate (EDMA) in the presence of a porogen in fused-silica capillaries of 100 mum I.D. The abnormal phenomenon that retention factors for neutral species decreases with applied voltage in CEC was observed. Capillary electrophoresis (CE) instruments usually require a period of time to increase voltage from 0 kV to desired value, which is called as ramp time. Such ramp time and any error in the determination of dead time should be taken into account during the accurate calculation of retention factors. After the correction of the retention factors, the plots of the corrected factors for alkylbenzene versus applied voltage were made, the absolute value of the plot slopes are less than 1.8 X 10(-4), Which indicates that the corrected retention times for neutral species do not show any dependence on applied voltage. Further, the plots of the corrected retention times for acidic and basic compounds versus the reciprocal of applied voltage were drawn, where the target compounds were eluted in neutral form. The very nice linearity of the plots was obtained. The linear correlation coefficients are over 0.999. Here, the slopes of the plots represent