The separation of biomolecules using capillary electrochromatography

The unique properties of capillary electrochromatography such as high performance, high selectivity, minimum consumption of both reagents and samples, and good compatibility with mass spectrometry make this technique an attractive one for the analysis of biomolecules including peptides, proteins, carbohydrates, nucleosides and nucleoticles. Irreversible adsorption between the biomolecules and the charged packing surface leads to a lack of reproducibility and serious peak tailing, so various approaches have been taken to overcome this and to improve the technique for future challenges.

Capillary array electrophoresis with confocal fluorescence rotary scanner

A capillary array electrophoresis system with rotary corifocal fluorescence scanner was reported. High speed direct current rotary motor combined with a rotary encoder and the reflection mirror has been designed to direct exactly the excitation laser beam. to the array of capillaries, which are symmetrically distributed around the motor. The rotary encoder is introduced to accurately orient the position of each capillary and its output signal triggers the data acquiring system to record. the fluorescence signal corresponding to each capillary. Separations of several amino acids are demonstrated by eight-channel capillary array electrophoresis built by ourselves.

Capillary electrochromatographic separation of ionizable compounds with a molecular imprinted monolithic cationic exchange column

A polymer-based monolithic capillary column imprinted with 4-aminopyridine (4-AP) was prepared by a thermally-initiated polymerization process; and its performance as a capillary electrochromatographic medium was evaluated in separating 4-AP and 2-AP isomers. The effects of experimental parameters, such as pH value and ionic strength of the buffer, the acetonitrile content in the mobile phase, and the applied voltage, on the resolution of these isomers had been carefully investigated. It was found that in the retention process there were interplays of multiple mechanisms of ion-exchange, molecular imprinting, and electrophoresis. These mechanisms allowed more sophisticated control of experimental parameters in the separation of ionizable compounds.

Preparation and characterization of monolithic columns for capillary electrochromatography with weak electroosmotic flow

Monolithic columns of capillary electrochromatography (CEC) with weak electroosmotic flow (EOF) have been prepared by in situ polymerization of butyl methacrylate and ethylene dimethacrylate, without any charged groups in the reaction mixture. The reproducibility of such columns has been proved good no matter whether they are prepared in the same batch or in different batches. In the case of BMA-EDMA monoliths, besides the traditional ternary mixture - 1-propanol, 1,4-butanediol, and water, binary porogenic solvents with only alcohols have also been adopted. Compared with ternary porogenic solvents, the design with binary ones allows for fine control of the pore diameter and the formation of the specific surface of the monolithic polymers. The composition of porogenic reagents has also been shown to have an effect on EOF in the column systems. In addition, the Joule heat effect in such columns has been studied by varying the inner diameter of columns. Through the separation of acidic compounds, monolithic columns with low EOF have shown potential in the analysis of charged samples.

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.

Capillary electrophoretic analysis of arsenic species with indirect laser induced fluorescence detection

The development of a method for determining arsenic species by capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence (LIF) is described in this paper. The buffer pH, the concentration of fluorescein, the nature and the concentration of the background electrolytes (BGEs) were defined. When 2.0 mM NaHCO3 (pH 9.28) with 10(-7) M fluorescein was used as the buffer, arsenite (As(lll), dimethylarsonic acid (DMA), monomethylarsonic acid (MMA), and arsenate (As(V)) were all separated from one another. The limits of detection for the four arsenic species were p p in the range of 0.12-0.54 mg/L. This method was used in the analysis of spiked arsenic species in tap and mineral water to demonstrate its usefulness. The results showed that both the recovery and the reproducibility of the developed method were acceptable.

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.