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.

Sieving matrices in capillary electrophoresis: inflection slope and double reciprocal plot.

We propose the use of a double reciprocal plot of the inflection slope and the concentration of a sieving polymer to compare sieving properties of polymers regardless of their concentration and to permit selection of appropriate sieving materials. Using this plot, the lines extrapolating the experimental values intersect the ordinate at the value corresponding to the inflection slope at the infinite concentration of the sieving polymer and the abscissa at the value characteristic for the given polymer. The latter corresponds to the polymer concentration at which the inflection slope equals half the inflection slope at an infinite polymer concentration. It is inversely proportional to intrinsic viscosity and this makes intrinsic viscosity an important physicochemical constant suitable for selection of new potent sieving polymers.

Galactomannans as a sieving matrix in capillary electrophoresis.

Purification of galactomannans including guaran, tara gum, and locust bean gum is described as well as their use as a sieving matrix in DNA sequencing by capillary electrophoresis (CE). Three methods of galactomannan purification were developed and tested using guaran. The first method is based on hydrolysis of proteins using alkali treatment and precipitation of guaran with acetone. The second method uses ion-exchange resins QAE Sephadex A-25 and SP Sephadex C-25 together with acetone precipitation. The third method is similar to the second one, except that it uses ion-exchange resins based on polystyrene, Source 30Q and Source 30S. Capillary zone electrophoresis of acetonitrile extracts from guaran revealed 4-5 characteristic major peaks and several minor peaks. Guar gum from different suppliers differed in the content of proteins. In purified guaran, protein peaks were detectable only using a 300-fold concentrate of extract. The content of proteins in the guaran purified using the third method was 0.001% m/m as determined by CE. The weight average molecular mass of purified guaran can be as large as 2.2 x 10(6). The purified galactomannans were used as a sieving matrix in DNA sequencing by CE. M13 DNA was sequenced to read lengths of about 600 bases in less than 90 min. Separation efficiencies exceeded 1 million theoretical plates for DNA fragments shorter than about 600 bases.

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.

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 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.

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.

Short tandem repeat typing by capillary array electrophoresis: comparison of sizing accuracy and precision using different buffer systems.

Polymorphic microsatellite markers are widely used in gene discovery and mapping, human identification, agricultural genetics, and diagnosis of triplet-repeat expansion disorders. Reliable genotyping of these markers requires polymerase chain reaction (PCR) amplification and very-high-resolution electrophoresis. Capillary array electrophoresis offers extremely fast, high-resolution separation of DNA and more automated sample processing because labor-intensive slab-gel pouring and sample loading are eliminated. We report a simple, reliable procedure for preparing PCR samples for electrokinetic injection into capillaries using a 96-well tray and float dialysis. We developed an improved sizing standard for genotyping and used it to evaluate systematically the sizing accuracy and precision of low-viscosity, replaceable matrix formulations. Our study sizing over 28,000 alleles yielded an average precision of +/- 0.12 bp for fragments up to 350 bp. Low-viscosity formulations permit low-pressure matrix injection (40 psi) and a turnaround time of 70 min for 48-96 samples.

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.

Versatile low-viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis.

The high-resolution separation of double-stranded DNA (dsDNA) has important applications in physical mapping strategies and in the analysis of polymerase chain reaction (PCR) products. Although high-resolution separations of dsDNA by capillary electrophoresis (CE) have been reported, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single formulation to separate a broad range (80 bp-40 kbp) of DNA fragments without the use of pulsed fields. We used a low-viscosity sieving medium (ca. 5 cP, at 25 degrees C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Mn 10(6)) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Mn 8 x 10(6)) to separate fragments between 1-40 kbp, within a single run. All PEO matrix formulations tested were compatible with a variety of intercalating dyes and with two different capillary wall coating methods. We obtained a detection limit of 25 fg of a 200 bp DNA quantitation standard using Vistra Green in the matrix. Resolution was best using short injection times (5 s or less) and low field strengths (approximately 100 V/cm). Sample runs were complete in 70 min, and use of the capillary array electrophoresis (CAE) system permitted high-throughput DNA analysis. The size range separated is approximately 10 times greater than with conventional slab gel separations.

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.

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.

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.

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.

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.

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.

Capillary zone electrophoresis of oligonucleotides. Factors affecting separation.

The influence of various parameters affecting separation of oligonucleotides by capillary zone electrophoresis has been examined. The effects of pH, ionic strength, and various additives, including highly charged cations such as spermine, were studied. Using polycytidines as model compounds, it was demonstrated that pH in the range of 5-8 and ionic strength in the range of 20-200 mmol/l do not influence the separation of oligonucleotides substantially. However, with the addition of spermine to the background electrolyte, migration order was inverted as the effective mobilities of the larger oligonucleotides were greatly decreased. With the addition of spermine and sodium dodecyl sulfate, the separation+ of these model oligonucleotides was also significantly affected. The best separation of a homologous series of polycytidines was obtained with a background electrolyte containing 60 mmol/l histidine, 30 mmol/l glutamic acid, 50 mmol/l sodium dodecyl sulfate and 3 mmol/l spermine.