Electrochromatography

Abstract: A microchip laboratory system and method provide fluid manipulations for a variety of applications, including sample injection for microchip chemical separations. The microchip is fabricated using standard photolithographic procedures and chemical wet etching, with the substrate and cover plate joined using direct bonding. Capillary electrophoresis and electrochromatography are performed in channels formed in the substrate. Analytes are loaded into a four-way intersection of channels by electrokinetically pumping the analyte through the intersection, followed by switching of the potentials to force an analyte plug into the separation channel.

Abstract: Publisher Summary Because the functional specificity of plasma lipoproteins resides in major part in the apolipoprotein moieties associated with the lipoproteins, techniques that combine size analysis, such as gradient gel electrophoresis, with apolipoprotein identification, either directly by immunoblotting or indirectly by immunoaffinity chromatography, appear highly promising for investigation of lipoprotein structure and function. This chapter discusses the application of gradient gel electrophoresis primarily to the analysis of HDL and LDL subpopulations and indicates approaches for more detailed identification of these subpopulations. Gradient gel electrophoresis entails the migration of charged particles through a matrix composed of increasing concentrations of polyacrylamide gel. The effective pore size of the matrix is progressively reduced as the gel concentration increases, resulting in differential retardation of the migrating charged particles. Although protein molecular weight determination is possible by gradient gel electrophoresis by use of appropriate calibration standards, such determination of lipoprotein molecular weights is still only approximate and requires validation by use of lipoprotein species with molecular weights determined by other methods.

Abstract: In electrochromatography, electrolyte is driven along a narrow bore chromatographic column at typical HPLC linear velocities by applying a potential gradient of around 50,000 V m−1. Contrasting with pressure driven LC, the tube bore is limited to not more than 200 µm by self heating. Thus miniaturisation is mandatory in electrochromatography whereas it is optional in pressure driven LC unless very small particles (e.g. 1 µm diameter) are used. Because the velocity profile in open tubular electrochromatography is close to that of perfect plug flow, contrasting with the parabolic flow profile in pressure driven LC, open tube electrochromatography provides plate efficiencies limited only by axial diffusion, and comparable to those obtainable in open tubular gas chromatography. Contrasting with pressure driven open tubular LC there is no requirement for the tube bore to be very small (e.g. <10 µm). Although pure electrophoresis is applicable only to ionised solutes, and is not strictly a chromatographic method, neutral species can be chromatographed by partitioning them between two phases which move at different rates, for example by using micellar solutions or colloidal sols as the moving fluid, thereby retaining the high plate efficiency of electrophoresis. With packed tubes electrochromatographic separations of high efficiency can be obtained with 5 µm particles and extremely high efficiencies should be obtainable without sacrifice of eluent velocity by using submicron particles. In principle, electrochromatographic methods can provide a wide range of partitioning methods of extremely high plate efficiency, and will, in future, undoubtedly rival conventional HPLC.

Abstract: Publisher Summary Disc electrophoresis has gained significant attention because of its well-defined, sharp separation boundaries, which can be achieved with only microgram quantities of protein. During disc electrophoresis a specific combination of ions forms a moving front of the “leading” ion followed by the “trailing” ion, thereby bracketing the sample protein between them. This permits concentration of the protein sample into an extremely sharp layer at the origin and yields the sharp resolution of individual boundaries. A further advantage of the method is offered by the use of polyacrylamide gel as anticonvection medium; the qualitative and quantitative selection of the gel components permits choice of a wide range of properties for the supporting medium. Thus, it is possible to anticipate the specific conditions for the best possible separation by proper selection of the gel components. Disc electrophoresis is also a powerful tool in establishing homogeneity of a protein although the results, as with all other methods used to establish purity of proteins, must be interpreted with caution. This chapter presents the electrophoretic procedure and methods of staining required to localize enzymes in gels.