Electromigration behavior of polysaccharides in capillary electrophoresis under pulsed-field conditions.

Various polysaccharides can successfully migrate through entangled polymer solutions during high-voltage capillary electrophoresis. For neutral polysaccharides, complexation with borate provides the electric charge needed for electromigration, while a fluorescent tag is needed to detect the solute bands with adequate sensitivity. At constant potentials between 50 and 300 V/cm, the charged polysaccharides undergo molecular stretching, resisting the desired separation according to their molecular mass. This problem can be overcome through the use of variable fields, pulsed along the separation capillary at a 180 degree angle. Variables of the pulsing experiment appear to have a profound influence on molecular shape rearrangements of polysaccharides with respect to the separation medium, as demonstrated here with highly efficient separations of polydextrans (8,000-2,000,000 Da).

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.

End-label, free-solution capillary electrophoresis of highly charged oligosaccharides.

The effect of fluorescent tags on the separation of negatively charged oligosaccharides, derived from a partially hydrolyzed kappa-carrageenan, was studied. When the charge-to-friction ratio of oligosaccharides is increased by the end-label, the migration order is from smaller to larger oligomers, and the resolution of larger oligomers could be improved by using a sieving medium. The migration order can be entirely reversed when the charge-to-friction ratio of the solute is decreased by the end-label. The experimental electrophoretic mobilities obtained in this work are in excellent agreement with the recently reported theoretical model (Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780). The maximum number of separated oligomers (Mmax) as a function of applied voltage and injection time was also studied, but no strong dependencies were found. Resolution between small oligomers could be significantly improved by following this separation principle.

Pulsed-field capillary electrophoresis: optimizing separation parameters with model mixtures of sulfonated polystyrenes.

The electrophoretic transport of high molecular weight charged solutes, both flexible and stiff polymers, has been studied by capillary electrophoresis under constant-field and pulsed-field conditions. Sulfonated polystyrenes were used as model solutes in different entangled polymer solutions. First, changes of the end-to-end distance vectors of flexible polymers were examined through the mobility/potential-gradient curves. Under pulsed-field conditions, the influence of different pulse shapes, frequencies, and amplitudes of forward and backward pulses on the electrophoretic mobilities of model solutes was studied. Resolution of the mixture components was strongly affected by changes in frequency of both sine-wave and square-wave pulses. The experimental results obtained under pulse-field conditions are roughly in agreement with the existing theories of electrophoretic transport.

Separation of large DNA fragments by capillary electrophoresis under pulsed-field conditions.

Capillary zone electrophoresis (CZE), using an entangled polyacrylamide solution, was applied to large DNA samples under pulsed-field conditions. Highly efficient separations were achieved under biased sinusoidal field and field-inversion pulsing regimes. The separations that were obtained with 8.3-48.5 kb lambda DNA standards and 48.5 kb-1 Mb lambda DNA concatamers (modified with ethidium bromide) clearly demonstrate a dramatic improvement in the separation time (roughly, 10-50 times) over the conventionally used slab-gel techniques. Moreover, the CZE method appears much more sensitive and amenable to component quantification and method automation.

High-performance capillary electrophoresis of glycoconjugates.

Capillary electrophoresis (CE) has recently emerged as a highly promising technique for the analysis and structural study of glycoconjugates. This article reviews the current status of CE and its potential in glycobiology. The separation media suitable for the analysis of monosaccharides, oligosaccharides and very large carbohydrate-based biomolecules are discussed. The detection aspects emphasize chemical derivatization approaches to tagging the glycoconjugates of interest for enhanced response in absorption and fluorescence spectroscopy.

End-label free-solution electrophoresis of the low molecular weight heparins.

The intact heparins are highly charged oligosaccharides. Their charge polydispersity and the possible occurrence of numerous isomers complicate the analysis of these biomedically important glycoconjugates. After unsuccessful attempts to resolve the low molecular weight heparins in entangled matrixes, or through the use of counterions (Stefansson, M.; Novotny, M. V. Anal. Chem. 1994, 66, 3466-3471), we have designed a unique end-label reagent to incorporate both a fluorescent moiety and a desirable frictional increment to the analyte molecules. The resolution of small oligomers was improved dramatically following this approach. We also propose a scheme, based on the end-label free-solution electrophoresis model (Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780), that could potentially predict the migration times of some oligomers of complex heparin mixtures.

Pressure refilled polyacrylamide columns for the separation of oligonucleotides by capillary electrophoresis.

The separation of oligonucleotides by capillary zone electrophoresis (CZE) was studied in fused silica separation capillaries filled by linear (noncrosslinked) polyacrylamide (PAA) solutions, introduced into the capillary from the stock by pressure after each analysis. The time-consuming in-capillary polymerization step could thus be avoided, and fast and reproducible repetition of the analyses was assured. The PAA concentrations varied within the range of 3-10% and both the reproducibility of the analyses and the stability of the solution in the capillary, with and without a chemically treated inner wall, were tested. Ferguson plots were used to assess the size selectivity of the separation.

The use of a bivalent counter anion to control the effective mobility of the hydrogen ion constituent in cationic isotachophoresis.

The use of a bivalent counter anion for cationic isotachophoresis was studied both theoretically and experimentally. A mathematical model was proposed and solved to give the effective mobility of the hydrogen ion constituent, uH,H, in the respective electrolyte systems. The theoretical values agreed well with the experimental data in a series of electrolyte systems. The dependence of uH,H on such parameters as pH of the leading electrolyte, ionic mobilities of leading and counter ions, and dissociation constants was calculated and discussed. A bivalent counter anion will prove useful for the separation of low mobility cations and weak bases, as shown for the separation of substituted anilines in a succinate electrolyte system.

High-Resolution Studies of Hyaluronic Acid Mixtures through Capillary Gel Electrophoresis.

Hyaluronic acid is a negatively charged polysaccharide with a high degree of polydispersity that makes the separation of its oligomers extremely difficult. Through the use of columns filled with a highly viscous polyacrylamide matrix, the unit resolution of hyaluronate oligomers could be achieved, up to at least 80 kDa of mass, through capillary electrophoresis. As analytical application examples, the fractions of enzymatically or ultrasonically degraded hyaluronates were monitored through this method. Because of the very high resolving power, peaks additional to the regular oligomers can be observed that are assumed to be conformers of this regular, unbranched biopolymer.

Step change of counterion--a new option in capillary zone electrophoresis.

In contrast to common capillary zone electrophoresis, with a constant composition of the background electrolyte, a new method is described, based on a change of the ionic matrix composition during the migration of the sample components. The proposed method expands the scale of pK's of analyzed compounds in a single run and affords new potential for optimizing the separation. The step change of the ionic matrix composition was achieved by displacing the primary matrix by the modified ionic matrix (electrolyte) which migrated against the movement of the sample components. The separation thus proceeded in the primary ionic matrix (electrolyte) while the detection was performed in the modified matrix with resultant shortening of the analysis time and convenient quantitation. A model mixture of five compounds, with a difference of four units in pK could be well resolved.

New block-copolymer thermoassociating matrices for DNA sequencing: effect of molecular structure on rheology and resolution.

A new family of matrices for DNA sequencing by capillary electrophoresis is presented. These matrices combine easy injection with high sieving performances, due to thermal switching between a low and a high viscosity state through a modest increase in temperature (approximately 20 degrees C). They are constructed from a hydrophilic polymer backbone with grafted lower critical solution temperature (LCST) side chains. The comb-like LCST copolymers are characterized in terms of size of the polymer backbone, the size of LCST side chains and the grafting densities. The dependance of rheological behavior and electrophoretic performance of these copolymers is correlated with their microstructure. Without complete optimization, a resolution of order 0.5, corresponding to a very reasonable limit for read length with current base calling softwares, could be achieved for segments around 800 bases differing by 1 base in less than one hour in a commercial ABI 310 apparatus.