Innovative developments in isotachophoresis (displacement electrophoresis)

This Mini Review is aimed at characterizing the innovative developments in isotachophoresis (ITP) during the past few years, discussing in turn new theoretical, analytical, preparative and applicative aspects of this unique separation method. Examples given from our laboratory include the study of the detailed dynamics of the ITP separation of four components by computer simulation and experimental validation in a capillary-type instrument with multiple sensors along the separation trough; the anionic ITP analysis in presence of a strong cathodic electroosmotic flow using an open-tubular fused-silica capillary with on-column multiwavelength detection, and the fractionation of proteins in a screen-segmented, rotating column as well as by recycling ITP.     

Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis)

The use of displacement electrophoresis (synonymous to isotachophoresis, steady-state stacking, and moving boundary electrophoresis) for recovery of DNA fragments from agarose and polyacrylamide gels is described. Complete recovery of DNA molecules ranging from oligonucleotides to 20 000-basepairs-long fragments was achieved. The DNA is recovered in a small volume (0.1-0.3 ml) and can be used directly in enzyme-mediated cleavage and ligation reactions. The recovered DNA contained no inhibitory contaminants as revealed by ligation or restriction enzyme cleavage.     

Precolumn concentration of protein samples by displacement electrophoresis (isotachophoresis) followed by high-performance molecular sieve chromatography on a packed agarose column

The use of displacement electrophoresis for the concentration of dilute protein solutions and the construction of a column suitable for this purpose are described. The concentrated protein zone can be pumped directly from the electrophoresis column into a gel-filtration column, which greatly reduces losses of protein. Recoveries of 95% or better were obtained even for small amounts of protein. The electrophoretically concentrated samples gave virtually the same elution profiles as did samples injected in a small volume without the use of electrophoretic preconcentration.     

Separation of hemagglutinin and neuraminidase from influenza virus membrane by column displacement electrophoresis (isotachophoresis) with preservation of their activities

Triton X-100-solubilized membrane glycoproteins (neuraminidase and hemagglutinin) from purified equine influenza virus particles were separated by column displacement electrophoresis (isotachophoresis) in the presence of Pharmalyte spacers. Electrophoresis was performed in a 1.80 cm glass electrophoresis column with Sephadex G-25 Fine serving as supporting medium. Triton X-100 was present in the system to suppress protein aggregation. Neuraminidase and hemagglutinin activities were preserved and appeared in the electropherogram as separate peaks with some overlapping.     

Synergism of capillary isotachophoresis and capillary zone electrophoresis

The combination of capillary isotachophoresis and capillary zone electrophoresis may enhance greatly the performance of analytical capillary electrophoresis with respect to both separation power and the concentration sensitivity. The concentrating effects and the separation power of isotachophoresis allow the analysis of diluted samples and the elimination of interferences due to bulk components. The separation process of zone electrophoresis enables one to resolve the stack of trace analytes and detect the resulting individual zones with high sensitivity. The transition of isotachophoresis into zone electrophoresis plays the key role in the overall performance of this hyphenated technique. This article describes the dynamics of the conversion of isotachophoresis into zone electrophoretic mode and shows that the key role is played by the segments of the leading and terminating zones from the isotachophoretic stage. The magnitude of these segments directly effects the detection time as well as the separation width of the peaks of analytes. It is shown that these effects are also important in the analyses by capillary zone electrophoresis where isotachophoresis is induced by the sample itself. Finally, the paper presents a list of recommended, user-friendly, electrolyte systems which enable one to simply predict the performance of the combination isotachophoresis-zone electrophoresis.     

Preparative capillary electrophoresis based on adsorption of the solutes (proteins) onto a moving blotting membrane as they migrate out of the capillary.

A micropreparative capillary electrophoresis apparatus equipped with a new type of fraction collection device is described: solutes, such as proteins, are adsorbed onto a moving blotting membrane (for instance a polyvinylidene difluoride membrane) as they migrate electrophoretically out of the capillary. The adsorbed proteins are visualized by conventional protein staining methods or by fluorescent labeling. Specific identification of separated components by an immunological technique is demonstrated. The method also offers the potential to analyze proteins and peptides collected on the membrane by gas phase sequencing and mass spectrometry.     

Counter-flow isotachophoresis on cellulose acetate membranes. Role of electroendosmosis]

A variant of counter-flow isotachophoresis of proteins on cellulose acetate membranes is proposed. The liquid counter-flow is created by electroendosmosis in the membrane. Proteins are concentrated at the Kolrausch boundary during isotachophoresis in the presence of ampholytes. The method permits one to make microanalysis of proteinic mixtures in diluted solutions, and it can be used in combination with immunodiffusion and immunoelectrophoretic methods of antigenic protein detection.     

Preparative electrophoresis: on the estimation of maximum temperature

The quantity of proteins processed by an electrophoretic technique is proportional to the cross-sectional area of the gel. For preparative purifications, an increase in the cross-sectional area is desired, but the Joule heating phenomenon restricts such an increase. The governing heat equation is analyzed and simplified with reference to Counteracting Chromatographic Electrophoresis. The application of the method of weighted residuals yields a compact and accurate solution for the maximum temperature rise in the column which is suitable for design calculations. Similar estimations indicate the efficiency of heat dissipation in annular configuration.List of Symbols C _p specific heat capacity, J g^–1 K^–1 - h heat transfer coefficient at the wall, W cm^–2K^–1 - i current density, A cm^–2 - k effective thermal conductivity of the packing, W cm^–1 K^–1 - k _b electrical conductivity of the buffer, mho cm^–1 - k _e effective electrical conductivity of the packing, mho cm^–1 - k _g electrical conductivity of the gel, mho cm^–1 - L length of the packing, cm - N _Pr Prandtl number - N _Re Reynolds number - r radial coordinate, cm - r _i inner radius of annulus, cm - r _o outer radius of annulus, cm - S heat source term, defined by eqn. (6) - T temperature, K - T _c cooling fluid temperature, K - T _i initial temperature, K - T _max highest temperature in the column, K - u superficial buffer velocity, cm s^–1 - V voltage gradient, V cm^–1 - porosity of the packing, dimensionless - buffer density, g cm^–3 - temperature, dimensionless Material presented in this paper has been adapted from the author's dissertation [15] which was accepted (supervisor: Dr. Jean B. Hunter) by the Cornell University Graduate Faculty in partial requirement of a graduate degree. Thoughtful discussions with Professors J. Robert Cooke and Michael L. Shuler regarding the annulus problem and the financial support provided by the Department of Agricultural and Biological Engineering, Cornell University, Ithaca, USA are gratefully appreciated.     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.