Capillary zone electrophoresis of serum proteins: study of separation variables.

Electrophoresis of serum proteins is one of the traditional applications of zone electrophoresis. Whereas electrophoresis in supporting media uses usually 5,5'-diethylbarbiturate at pH 8.6 as the buffer, in capillary zone electrophoresis with on-line UV detection, this electrolyte is of little use because of its high UV absorbance. For that reason, a number of operational electrolytes differing in composition were tested for use in capillary electrophoresis of serum proteins. The influence of the pK(A) of co-ions and counter ions and the concentration of the operational electrolyte was examined. If 0.1 M methylglucamine-0.1 M epsilon-aminocaproic acid or 0.1 M methylglucamine, -0.1 M gamma-aminobutyric acid is used as the operational electrolyte, capillary electrophoresis separates serum proteins into more than ten zones.

Selectivity, differential mobility and resolution as parameters to optimize capillary electrophoretic separation.

Selectivity, differential mobility, and resolution have been tested as the optimization functions to find the optimum pH of operational electrolyte for separation by capillary electrophoresis when organic acids occurring in human serum have been selected as a model mixture for computer simulations. Using tabulated values of ionic mobilities and pKa values, either selectivity or differential mobility or resolution for the hardest-to-separate pair of separands are calculated and plotted vs. pH. The optimum pH is the pH value, at which the optimization function reaches its maximum.

Capillary zone electrophoresis of organic acids in serum of critically ill children.

Capillary zone electrophoresis with indirect UV detection was found to be suitable for the determination of organic acids in serum. Serum can be analysed directly without any deproteination in a capillary coated with linear polyacrylamide. With 10 mM epsilon-aminocaproic acid-10 mM mandelic acid (pH 3.8) as the operational electrolyte, anions such as pyruvate, phosphate, citrate, malate, acetoacetate and lactate can be determined in 12 min. In quantitative analysis, the calibration line for lactate is linear over the range 0-10 mM. The detection limit for citrate was 8 microM. The effect of the chloride concentration on the migration times of minor peaks is discussed. The potential of the method was demonstrated by analysing sera from several critically ill children.

DNA sequencing by capillary electrophoresis (review).

DNA sequencing by capillary electrophoresis has been reviewed with an emphasis on progress during the last four years. The effects of sample purification, composition of sieving matrices, electric field strength, temperature, wall coating and DNA labeling on the DNA sequencing performance are discussed. Multicapillary array instrumentation is compared with one-capillary systems. Integrated systems that perform the whole DNA sequencing operation online starting from the DNA amplification through base calling and data processing are discussed.

Physico-chemical properties of interferon produced by a mixed leukocyte suspension.

Physico-chemical properties of partially purified interferon produced by a mixed culture of human peripheral blood leukocytes following induction with double-stranded RNA extracted from f2 phage infected Escherichia coli were studied. Molecular heterogeneity of the interferon preparation was demonstrated by gel chromatography on a Sephadex G-100 column, disc electrophoresis in polyacrylamide gel and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The first two methods revealed 5 peaks, the latter 7 peaks of interferon activity. There was no difference in the molecular nature of interferon produced by cultures exposed to the inducer for the whole period of incubation and that produced by cultures from which the inducer was removed after a short induction time.

Ability of human polymorhonuclear blood cells to produce interferon after induction with phage double-stranded RNA.

The ability of human peripheral blood leukocytes to produce interferon in response to phage double-stranded (ds) RNA was studied. Under the conditions used, interferon was produced not only by lymphocytes but also by polymorphs and monocytes. These "pure" cultures showed no marked differences in the degree of interferon production as compared with the mixed leukocyte culture. The involvement of polymorphs in the production of interferon induced by phage ds RNA is discussed.

High-throughput DNA sequencing on a capillary array electrophoresis system.

A capillary array electrophoresis apparatus capable of running and analyzing 48 DNA sequencing samples simultaneously has been constructed. The instrument uses a replaceable sieving buffer and incorporates a convenient method for introducing the buffer into the capillaries. Data from laser-induced fluorescence are collected as four separate images, one for each optical channel. The integrated data analysis software employs an open architecture that allows use of any DNA base-calling algorithm. DNA sequencing runs are completed in approx. 1 hr (approximately 500 bases), and instrument turnaround time between runs is less than 15 min. Overall, the instrument throughput is on the order of 720 templates/day, or 360,000 bases/day.

Capillary zone electrophoresis of proteins.

This review article with 237 references is focused on capillary zone electrophoresis (CZE) of proteins. It includes discussion of modeling electrophoretic migration of proteins, sample pretreatment before the analysis, methods reducing the sorptions of proteins on the capillary wall, and techniques for increasing selectivity by using electrolyte additives including the sieving matrices. Significant progress in detection techniques, namely in laser-induced fluorescence and mass spectrometry, is emphasized. Modifications of CZE using specific interactions, such as affinity capillary electrophoresis or capillary immunoelectrophoresis, are debated as well as combination of CZE with other separation methods such as high performance liquid chromatography (HPLC). A number of practical applications of CZE of proteins are described.

Recent developments in capillary zone electrophoresis of proteins.

This review article with 125 references describes recent developments in capillary zone electrophoresis of proteins. It encompasses approximately the last two years, from the previous review (V. Dolník, Electrophoresis 1997, 18, 2353-2361) through Spring 1999. Topics covered include modeling of the electrophoretic properties of proteins, sample preconcentration and derivatization, wall coatings, improving selectivity, special detection techniques, and applications.

Polymer wall coatings for capillary electrophoresis.

This review article describes the preparation of dynamic and static polymeric wall coatings for capillary electrophoresis. Properties of bare fused-silica surfaces and methods for the characterization of capillary coatings are summarized. The preparation and basic properties of neutral and charged wall coatings are considered. Finally, advantages and potential applications of various coatings are discussed.

Determination of oxalate in human serum by multicolumn isotachophoresis.

The practical usefulness of multicolumn isotachophoresis was demonstrated by the determination of oxalate levels in human serum. 10 mmol/L HCl and 100 mmol/L Na2HPO4 served as the leading and terminating electrolyte, respectively. For the stabilization of the isotachophoretic zones in large cross section channels a suspension of Bio-Gel P-300 in the leading electrolyte was used. For the analysis ca. 1 mL of fresh serum was required and 1.2 mL of 1:1 mixture of serum and the suspension of Bio-Gel P-300 in deionized water was applied as sample. The time of analysis ranged between 45-49 min. The detection limit of analysis was determined to be 50 nmol/L. Reproducibility of analysis of 1 mumol/L oxalate in water standard was found to be 2.6% (n = 6).

Large sample volume preseparation for trace analysis in isotachophoresis.

An isotachophoretic device for the analysis of trace components in a sample with the efficient pre-separation and elimination of bulk components has been suggested and realized. A large volume of the sample in question is separated in a rectangular wide-bore channel packed with a suspension of a granulated polyacrylamide gel and analyzed in free electrolytes in a narrow-bore tube. Trace components, the concentrations of which are 10(-6) mol/l, can be analyzed in 60-70 min applying ca. 1 ml of the sample even in the presence of bulk excess of a major component the concentration of which is by 5 orders of magnitude higher.

Electromigration behavior of poly-(L-glutamate) conformers in concentrated polyacrylamide gels.

During electrophoretic separation of anionic polyamino acids, resolution according to the number of peptide units can be achieved in capillaries filled with hydrophilic gels. While polyaspartate preparations yield single peaks for the individual oligomers at pH above 8.0, polyglutamates exhibit an anomalous behavior of peak splitting, which is attributed here to the separation of the oligopeptide conformers. An Asp-Glu (1:1) copolymer yields single peaks under similar conditions. At pH near 4.5, where polyglutamate is expected to exist in its alpha-helix form, peak splitting disappears. Upon heating to 95 degrees C for at least 120 h (procedure described to transform the alpha-helix into a beta form), peak splitting disappeared, but could be reestablished after cooling for several days. When a highly charged cation spermine was added to the operational electrolyte, triple peaks appeared in the electropherogram due to the ion-pair formation. The largest peak in every triplet has been tentatively assigned to the alpha-helix form. The electrophoretic results described have been largely supported by CD spectra.

Capillary electrophoresis of proteins 1999-2001.

This review article with 223 references describes recent developments in capillary electrophoresis (CE) of proteins and covers papers published during last two years, from the previous review (V. Dolnik, Electrophoresis 1999, 20, 3106-3115) through Spring 2001. It describes the topics related to CE of proteins including modeling of the electrophoretic properties of proteins, sample pretreatment, wall coatings, improving selectivity, detection, special electrophoretic techniques, and applications.

Separation of amino acid homopolymers by capillary gel electrophoresis.

Gel-filled capillaries utilizing highly concentrated and moderately cross-linked acrylamide-type gels in capillary electrophoresis were successfully applied to the separation of the individual oligomers of various poly(amino acids). Mixtures of both anionic and cationic nature were adequately resolved. While UV detection at 220 nm was mostly utilized, the polyanions with N-terminal groups can also be tagged with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) for a more sensitive detection by a laser-induced fluorescence detector.

Capillary electrophoresis on microchip.

Capillary electrophoresis and related techniques on microchips have made great strides in recent years. This review concentrates on progress in capillary zone electrophoresis, but also covers other capillary techniques such as isoelectric focusing, isotachophoresis, free flow electrophoresis, and micellar electrokinetic chromatography. The material and technologies used to prepare microchips, microchip designs, channel geometries, sample manipulation and derivatization, detection, and applications of capillary electrophoresis to microchips are discussed. The progress in separation of nucleic acids and proteins is particularly emphasized.

Capillary electrophoresis in sieving matrices: selectivity per base, mobility slope, and inflection slope.

We compare the migration behavior of DNA sequencing fragments in hydroxyethyl cellulose (HEC) to the theoretical model of migration in the reptation mode. Good agreement was found for the mobility curve. We derived empirical equations for the relationship between selectivity per base and sieving matrix concentration and between the mobility slope and matrix concentration. We propose the inflection slope, i.e., the slope of the log-log mobility curve at its inflection point, as the quantitative parameter of sieving performance.

Recent developments in isotachophoresis.

This paper is summarizing the contributions to the analytical capillary isotachophoresis published during the period 1981-1984. It characterizes the present state of the method and covers theory, fundamental analytical aspects, instrumentation and applications. Special attention was payed to the fundamental analytical aspects, and a detailed discussion is given of the selection of electrolyte systems, stability of zones and separability of substances. The present commercial instrumentation is also briefly described.

Experimental evaluation of the separation efficiency in capillary electrophoresis using open tubular and gel-filled columns.

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.

Electrophoretic separations of proteins in capillaries with hydrolytically stable surface structures.

A procedure for obtaining highly stable coated capillaries for use in capillary electrophoresis (CE) is described. Reaction of surface-chlorinated fused silica capillaries with the Grignard reagent, vinyl magnesium bromide, followed by reaction of the vinyl group with acrylamide, results in an immobilized layer of polyacrylamide attached through hydrolytically stable Si-C bonds. This method is an extension of the capillary coating procedure described previously by Hjerten, differing in the means by which the polyacrylamide layer is bonded to the capillary walls. Capillaries treated in the manner described here can be used over a pH range of 2-10.5, without noticeable decomposition of the coating. In comparison to uncoated capillaries, separations of proteins using such coated capillaries are improved due to a reduction in protein adsorption to the capillary walls, although interaction is still present to some degree as evidenced by an inability to obtain plate counts as high as those predicted by theory. Electroosmotic flow is virtually eliminated in the coated capillaries, resulting in improved reproducibilities of protein migration times in comparison to uncoated capillaries. Additionally, peak skew is evaluated for model proteins and improvements are noted for the coated capillaries. Results are presented for separations of model protein mixtures, comparing the performance of the vinyl-bound polyacrylamide coated capillaries and uncoated capillaries at both high and low pH extremes.