Influence of operating parameters on reproducibility in capillary electrophoresis.

The reproducibility of two migration parameters (retention time and mobility) of a seven-component test mixture was examined under various operating conditions using laboratory-built capillary electrophoresis systems. It was found that the frequency of rinsing the capillary and the solutions used for rinsing had the greatest effect on migration reproducibility. In addition, it was found that the migration behavior of solutes that interact with micelles is not repeatable unless the proper rinse protocol is applied. Inconsistent migration behavior is linked to inconsistent total current of the system. Preliminary investigations indicate that the fluctuation in total current were associated with non-equilibrium conditions between the buffer and the capillary wall.     

How to achieve higher repeatability and reproducibility in capillary electrophoresis.

The influence of experimental parameters on precision of migration times and accuracy of integration has been studied. The repeatability of migration times strongly depends on the proper selection of the rinse steps between each run of a CE system. The rinse steps have to be optimized additionally with each separation system. This is especially important for systems providing an anodic electroosmotic flow. The errors introduced by the integration software were studied by transferring the same data sets to different commercial available integration softwares. A strategy for the transfer of raw data files between several softwares is described. By using systems with identical peak areas or with identical peak heights it could be shown that the newly introduced softwares can cope better with the leading or tailing peaks. The RSD of quantitation strongly depends on the signal-to-noise ratio. At S/N ratios larger than 35 no differences between the various softwares studied could be observed. At lower S/N ratios the newly released softwares are to be preferred.     

Factors affecting selectivity of inorganic anions in capillary electrophoresis

Capillary zone electrophoretic separations of inorganic anions are largely governed by the intrinsic (infinite dilution) mobility of the anion. This in turn is a function of the hydrodynamic friction caused by the size of the ion and the dielectric friction caused by the charge density of the anion re-orienting the surrounding solvent. The influence of these factors on the mobility of anions is examined in both water and nonaqueous solvents. The influence of other experimental parameters, such as ionic strength, ion association, electroosmotic flow modifier concentration, and the addition of complexing agents such as polymeric cations, cyclodextrins, crown ethers and cryptands are also reviewed. From this discussion, some rules of thumb as to when different approaches will be most effective are drawn.     

Interlaboratory reproducibility of mobility parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

An interlaboratory pilot study was performed to determine the reproducibility of mobility parameters in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The study was performed by an intended small number of laboratories (three) that used different brands of instruments (two). The effective mobility was corrected using standards by a method that was recently introduced to obtain a more reproducible migration parameter. A test set of 20 acidic test compounds and 5 reference compounds were analyzed during five days in each laboratory using CZE and MEKC. Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). Analyses were carried out using fused-silica capillaries at an electric field strength of either 52.6 kV/m or 37.5 kV/m. The interlaboratory reproducibility (mean RSD) of the effective mobility was 3.0% for CZE and 6.7% for MEKC. After applying the correction method, these values became 3.0% for CZE and 3.3% for MEKC, which is adequate for systematic toxicological analysis (STA) applications. A significant improvement of reproducibility for the calculated corrected effective mobility mu(eff)c was observed when variations are high. Therefore, it is recommended to use the correction method in interlaboratory situations, especially when instruments and capillaries from different manufacturers are used.     

Parameters influencing separation and detection of anions by capillary electrophoresis

Electrolyte composition is critical in optimizing separation and detection of ions by capillary electrophoresis. The parameters which must be considered when designing an electrolyte system for capillary electrophoresis include electrophoretic mobility of electrolyte constituents and analytes, detection mode, and compatibility of electrolyte constituents with one another. An electrolyte system based on pyromellitic acid is well suited for use with indirect photometric detection, and provides excellent separations of anions. The ability to modify the electrophoretic mobility of pyromellitic acid as a function of ph provides flexibility in matching electrophoretic mobilities of analytes. Additionally, the use of alkyl amines as electroosmotic flow modifiers allows the rapid separation of anions by reversing the direction of electroosmotic flow in a fused-silica capillary. The optimization of a capillary electrophoresis electrolyte for anion analysis is also discussed in terms of pH, ionic strength and applied voltage. The effect of organic solvent on separation selectivity is also discussed.     

Improvement of reproducibility in capillary zone electrophoresis by sequential application of high voltage and pressure

In capillary zone electrophoresis (CZE) problems arise concerning the reproducibility of the measurements. This is due to variations in electroosmotic flow and analyte velocities. The discontinuous use of high voltage for the separation of analytes and the pressure for the transport to the detector offers the possibility to suppress the main causes for these phenomena. The procedure is demonstrated for the separation of 4 phenol derivatives. An increase in reproducibility is found for standard solutions from about 3% to 1%.     

Factors affecting the separation of mandelic acid enantiomers by capillary electrophoresis

Chromatographia - The separation of the optical isomers of mandelic acid by capillary electrophoresis using γ-cyclodextrin as a chiral selector is presented. The mobility of the enantiomers...     

Factors affecting the capillary electrophoresis of ricin, a toxic glycoprotein

Conditions for the analysis of ricin with capillary electrophoresis were investigated. Uncoated and coated columns were tested with a variety of different buffer combinations which included different principal components, pH, ionic strength, and additives. Of the combinations tested, uncoated columns used with either zwitterionic salts or putrescine gave the best results. Multiple peaks were resolved with these conditions. Coated columns generally yielded between 1000 and 5000 plates with several buffer combinations. Ricin may be analyzed faster and with greater resolution with capillary electrophoresis employing untreated fused-silica columns than by using other chromatographic techniques.     

Improving the reproducibility in capillary electrophoresis by incorporating current drift in mobility and peak area calculations

The traditional way of calculating mobility and peak areas in capillary electrophoresis does not take into account the changes in the buffer viscosity at different thermostatic control and that the analytes may accelerate during the individual runs due to Joule heating effects. We present a method for accounting for these changes based on the monitored changes in current during the separation. The calculation method requires measuring the initial resistance of the buffer filled capillary, performed using a 0.2 min voltage ramping at the start of a separation. The mobility calculation corrected for current drift allowed identification of the tested analytes independent from capillary dimensions, electric field strengths and temperature control. Furthermore, the peak areas become less influenced by the experimental conditions, since the velocities of the analytes passing the detector are corrected for the acceleration during the run. The short voltage ramping could be further used to evaluate the heat transfer of the capillary to the surroundings and to estimate the temperature changes during the separation. The temperature was shown to change the ionization of 2-phenylethylamine in accordance to a pKa dependency of primary amines reported in literature.     

Intra- and interinstrument reproducibility of migration parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

The intra- and interinstrument reproducibilities of four capillary electrophoresis instruments were studied for identification purposes in systematic toxicological analysis (STA). A test set of 20 acidic test compounds and 5 reference compounds were analyzed for five days on each instrument using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The buffers consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate and 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries at an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA. To deal with the poor reproducibility of the migration time, we recently introduced the corrected effective mobility. In this study, we investigated the intra- and interinstrument reproducibility of the migration time, the effective mobility, and the corrected effective mobility. Large differences in intra-instrument reproducibility were found when the migration time was used. The calculation of the effective mobility and the corrected effective mobility diminished these differences and enhanced the interinstrument reproducibility roughly by a factor 3. For (corrected) effective mobilities, intrainstrument reproducibilities were between 0.8-2.6% and interinstrument reproducibilities were between 3.2-3.9%.     

Carbon nanotubes in capillary electrophoresis, capillary electrochromatography and microchip electrophoresis

Carbon nanotubes are among the plethora of novel nanostructures developed since the 1980s. Nanotubes have attracted considerable interest by the scientific community thanks to their extraordinary physical and chemical properties. Research areas have flourished in recent years and now include the nano-electronic, (bio)sensor and analytical field along with many others. This review covers applications of carbon nanotubes in capillary electrophoresis, capillary electrochromatography and microchip electrophoresis. First, carbon nanotubes and a range of electrophoretic techniques are briefly introduced and key references are mentioned. Next, a comprehensive survey of achievements in the field is presented and critically assessed. The merits and downsides of carbon nanotube addition to the various capillary electrophoretic modes are addressed. The different schemes for fabricating electrochromatographic stationary phases based on carbon nanotubes are discussed. Finally, some future perspectives are offered.      

Non-equilibrium capillary electrophoresis of equilibrium mixtures--appreciation of kinetics in capillary electrophoresis

We describe a new electrophoretic method (patent pending), Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM), and demonstrate its application to the study of protein-DNA interactions. A single NECEEM experiment allows for the determination of equilibrium and kinetic parameters of protein-DNA complex formation. The equilibrium mixture is prepared by mixing protein and DNA; it contains three components: free protein, free DNA, and the protein-DNA complex. A small plug of such a mixture is injected onto a capillary and the three components are separated under non-equilibrium conditions using a run buffer that does not contain the components of the equilibrium mixture. The protein-DNA complex decays during the NECEEM separation; the resulting electropherograms contain characteristic peaks and exponential curves. A simple analysis of a single electropherogram reveals two parameters: the equilibrium dissociation constant of the protein-DNA complex and the monomolecular rate constant of complex decay. The bimolecular rate constant of complex formation can then be calculated as the ratio of the two experimentally-determined constants. NECEEM was applied to find the equilibrium and kinetic parameters of interaction between an E. coli single-stranded DNA binding protein and a fluorescently-labeled oligonucleotide. The constants determined by NECEEM are in good agreement with those obtained by other methods. The new method is simple, fast, and accurate. It can be equally applied to other non-covalent molecular complexes.     

Thermostating in capillary electrophoresis

Summary The use of high voltages across a electrophoresis capillary will increase the temperature of the buffer due to Joule heating. As a result temperature control in CE is rather important since variations in the buffer temperature will result in changes in the pH of the buffer, peak shape, migration time, reproducibility, efficiency, 3-D structure of macromolecular analytes, etc. Six different thermostating systems have been evaluated: (i) natural convection, (ii) fan, (iii) home-made and (iv and v) two commercially available high-speed air and a (vi) liquid thermostated device. In all cases the temperature of the buffer in the capillary is calculated according to the temperature-conductivity relationship. For this purpose two parameters are introduced describing temperature control: the temperature onset (δT) and the temperature rise factor (α). From these results, it can be concluded that high speed air thermostating can be as efficient as liquid thermostating.     

Detection in capillary electrophoresis

A review of the four major, on-line, capillary electrophoresis (CE) detection modalities is presented. It is shown that each detection method, fluorescence, absorbance (conventional and nonconventional), electrochemical and refractive index, have distinct advantages and limitations when applied to analysis in a CE format. Various aspects of CE detection are considered and a perspective regarding the applicability of the technique is provided. It is shown that because of widely varying detection limits (ranging from single molecule to 10(-5) M) and detection scheme complexity, the particular application should dictate the selection of detection methodology in CE.     

Chiral capillary electrophoresis: facts and fiction on the reproducibility of resolution with randomly substituted cyclodextrins

Comparative enantioseparation of the enantiomers of 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate was performed with cyclodextrin (CD)-modified capillary electrophoresis (CE). Two single isomers, beta-CD, heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD), and heptakis(2,6-di-O-methyl)-beta-CD (DM-beta-CD) of 98% purity as well as heptakis(2,3-di-O-acetyl)-beta-CD were used and compared in terms of resolution power to randomly methylated and corresponding acetylated beta-CDs, which were synthesized in our laboratory. The methylated ones were characterized by means of matrix-assisted laser desorption/ionization-time of flight-(MALDI-TOF) mass spectrometry. By testing defined mixtures of single isomers and comparing their resolution power to randomly substituted CDs of similar degree of substitution we could show, that a simple characterization by the average molecular degree of substitution (DS) is not sufficient. In order to get reproducible results, a clearly defined substitution pattern is necessary, which is not given using randomly substituted CDs. Taken together, a validation of a chiral separation with "undefined" CD derivatives is almost impossible.     

Factors affecting reproducibility in the manufacture of glass and siliceous glass open tubular (capillary) columns

Summary Column characteristics affecting the chromatographic behavior of glass and siliceous glass (fused silica) capillary columns include the dimensional uniformity of the column, the physical and chemical characteristics of the column wall and the characteristics of the liquid phase. In the case of the coated column the uniformity and thickness of the liquid phase film are the most important criteria affecting column reproducibility. The paper discusses these factors and their influence on column performance.Presented in part at the Symposium on Standardized Materials for Chromatography, 181 st National American Chemical Society Meeting, Atlanta, Georgia, March 29–April 3, 1981.     

Factors affecting the reproducibility and reliability of retention simulation in any form of temperature programmed capillary GC

Conversion of Kováts retention indices on a given stationary phase into the thermodynamic parameters of compounds on a given column leads to a simplified method for retention simulation in isothermal, linear, and multi-ramp temperature programmed capillary gas chromatography. The influence of numerical methods used in the computation, the temperature coefficient of Kováts indices, and the experimental factors such as isothermal temperatures selected in the measurement of n-alkanes, column characterization and sample overloading, on the reproducibility and accuracy of simulation were discussed and examined. When the column used is properly characterized, the error between the simulated values and the experimental data is within ± 0.5 index unit or less than ± 1% of retention time.     

Cyclic capillary electrophoresis

A strategy is described here for increasing both the resolution and the flexibility of capillary electrophoresis performed in a sieving medium of ungelled polymer. This strategy is based on analysis and, sometimes, re-analysis that is done in several stages of constant-field electrophoresis. Enhancement-stages are between the analysis-stages. An enhancement-stage (i) increases the separation between peaks, while (ii) moving DNA molecules in the reverse direction. An enhancement-stage is based on an electrophoretic ratchet generated by a pulsed electrical field that can be zero-integrated. The ratchet-generating pulses are longer than the field pulses that have previously been used to improve the resolution of DNA molecules. No limit has been found to the resolution enhancement achievable. Apparently, diffusion-induced peak broadening is inhibited and, in some cases, may be reversed by the ratchet. The enhancement-stages are critically dependent on the electrical field-dependence of a plot of electrophoretic mobility as a function of DNA length. To generate the pulsed electrical field, a computer-controlled system with a time resolution of 30 microseconds has been developed. Programming is flexible enough to embed other pulses within ratchet-generating pulses. These other pulses can be either the previously used, shorter field-inversion pulses or high-frequency periodic oscillations previously found to sharpen peaks.     

Ligand-exchange capillary electrophoresis

The fundamentals of ligand-exchange capillary electrophoresis are discussed, and the potential of the method in solving major problems, such as the separation of enantiomers and the determination of biologically active compounds poorly absorbing in the UV region (sugars, amines, and amino acids) is considered.     

Non-aqueous capillary electrophoresis

The benefits of non-aqueous capillary electrophoresis have been described in a number of recent publications. The wide selection of organic solvents, with their very different physicochemical properties, broadens our scope to manipulate separation selectivity. The lower currents present in non-aqueous solvents allow the use of high electric field strengths and wide bore capillaries, the latter in turn allowing larger sample load. In many cases detection sensitivity can also be enhanced. The potential of non-aqueous capillary electrophoresis is discussed throughout the paper, and the feasibility of capillary electrophoresis under non-aqueous media is demonstrated with reference to several applications.