Continuous zone electrophoresis

Proteins can ba fractionated by electrophoretic techniques on the basis of one or a combination of their three major properties size, net charge and relative hydrophobicity. Electrophoresis under native conditions is ideal for soluble proteins, where biological properties can often be retained. In contrast, more vigorous and often denaturing conditions must be used for analysis of less soluble proteins. Electrophoretic separations can be carried out using either a continuous or discontinuous (Multiphasic) buffer system. The techniques are referred to as continuous zone electrophoresis (CZE) or discontinuous ("disc") electrophoresis (also known as multiphasic zone electrophoresis, MZE). 

Example II Continuous Sample Preparation. For the primary sample preparation steps in a TAS, continuous techniques are clearly advantageous, e.g., filtration, dialysis, SPLITT [39] or free-flow electrophoresis (FFE) [40]. A special version of continuous zone electrophoresis in quartz channel structures has been presented by Mesaros et al. 

Fig. 9. Diagram representing the principle of the continuous zone electrophoresis apparatus of Svensson and Brattsten (1949).

In continuous-flow zone electrophoresis the solute mixture to be separated is injec ted continuously as a narrow source within a body of carrier fluid flowing between two electrodes. As the solute mixture passes through the transverse field, individual components migrate sideways to produce zones which can then be taken off separately downstream as purified fractions. 

Moseley, M. A., Deterding, L. J., Tomer, K. B., and Jorgenson, J. W. Coupling of capillary zone electrophoresis and capillary liquid chromatography with coaxial continuous-flow fast atom bombardment tandem sector mass spectrometry, /. Chromatogr., 480, 197, 1989. 

Kasicka, V., Pruslk, Z., Sazelova, P., Jiracek, J. and Barth, T., Theory of the correlation between capillary and free-flow zone electrophoresis and its use for the conversion of analytical capillary separations to continuous free-flow preparative processes. Application to analysis and preparation of fragments of insulin, ]. Chromatogr. A, 796, 211, 1998. 

Capillary zone electrophoresis coupled with fast cyclic voltammetric detection was developed by Zhou et al. [27] for the separation and determination of OTC, TC, and CTC antibiotics. All compounds were well separated by optimization of pH and complexation with a boric acid sodium tetraborate buffer. The detection limit using fast on-line cyclic voltammetric detection with Hg-film-microm electrode was 1.5 x 10-6 mol/L for OTC (signal to noise ratio > 2). A continuous flow manifold coupled on-line to a capillary electrophoresis system was developed by Nozal et al. [28] for determining the trace levels of OTC, TC, and DC in surface water samples. 

DGE a AC AMS APCI API AP-MALDI APPI ASAP BIRD c CAD CE CF CF-FAB Cl CID cw CZE Da DAPCI DART DC DE DESI DIOS DTIMS EC ECD El ELDI EM ESI ETD eV f FAB FAIMS FD FI FT FTICR two-dimensional gel electrophoresis atto, 10 18 alternating current accelerator mass spectrometry atmospheric pressure chemical ionization atmospheric pressure ionization atmospheric pressure matrix-assisted laser desorption/ionization atmospheric pressure photoionization atmospheric-pressure solids analysis probe blackbody infrared radiative dissociation centi, 10-2 collision-activated dissociation capillary electrophoresis continuous flow continuous flow fast atom bombardment chemical ionization collision-induced dissociation continuous wave capillary zone electrophoresis dalton desorption atmospheric pressure chemical ionization direct analysis in real time direct current delayed extraction desorption electrospray ionization desorption/ionization on silicon drift tube ion mobility spectrometry electrochromatography electron capture dissociation electron ionization electrospray-assisted laser desorption/ionization electron multiplier electrospray ionization electron transfer dissociation electron volt femto, 1CT15 fast atom bombardment field asymmetric waveform ion mobility spectrometry field desorption field ionization Fourier transform Fourier transform ion cyclotron resonance... 

Fig. 3.161. (A) Zone electrophoresis patterns of FITC-labelled transferrin samples by fluorescence detection. The unbound dye (providing a main peak and several minor ones) was not removed from the samples. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length 41 cm) X 75 pm i.d. injection of samples 100 mbar x s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut-off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 13 pm (1 mg/ml) Tf and (a) 0.01 mM FITC, (b) 0.1 mM FITC, and 1 mM FITC. (B) Zone electrophoresis patterns of an FITC-labelled transferrin sample by simultaneous fluorescence (upper trace, left axis) and UV detection (lower trace, right axis). The unbound dye shows several peaks with both detections. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length fluorescence 41 cm, UV 50.5 cm) X 75 pm i.d. injection of samples 100 mbar X s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 6.5 pm (0.5 mg/ml) Tf and 0.1 mM FITC. Reprinted with permission from T. Konecsni et al. [199].

The development of electrophoretic techniques afforded possibilities for fractionations based on charge density differences. Duxbury (1989) has reviewed applications of different electrophoretic separation methods, including zone electrophoresis, moving boundary electrophoresis, isotachophoresis, and isoelectric focusing (IEF). Preparative column electrophoresis (Clapp, 1957) and continuous flow paper electrophoresis (Hayes, 1960 summarized by Hayes et al., 1985) methods have been used to separate components isolated from sapric histosol soils. These techniques allowed separation of polysaccharides from the colored components the electrophoretograms of the colored components were diffuse, showing a continuum of components of different charge densities. 

An important advantage of two-dimensional methods is the solitary migration of each fraction on its own path. In contrast with unidimensional or zone electrophoresis where each fraction migrates its own distance but over a common path, no contamination of fractions is possible (Fig. 40). If the substrate is sufficiently long and wide and a high intensity electrical field is applied, it becomes possible to separate fractions reasonably completely from each other. If the physical forces involved are deployed skillfully, the electrical field can be driven up so that rapid and wide separations are obtained. This point is especially important in star electrophoresis, whereas in collecting electrophoresis mastery of the physical forces provides an excellent continuous preparative tool. 

The most common classification scheme in electrophoresis focuses on the nature of electrolyte system. Using this scheme, electrophoretic modes are classified as continuous or discontinuous systems. Within these groupings the methods may be further divided on the basis of constancy of the electrolyte if the composition of the background electrolyte is constant as in capillary zone electrophoresis, the result is a kinetic process. If the composition of the electrolyte is not constant, as in isoelectric focusing, the result is a steady-state process. 

Many 2D planar structures have been used to implement deflection (continuous flow) electrophoresis. The primary requirement is that flow and electrophoresis be carried out simultaneously and uniformly. Hanging paper curtains soaked with electrolyte and fed a stream of electrolyte from above served admirably for this purpose when the technique was initiated in the 1950s. In recent years thin flow channels enclosed between flat plates have become important. The process is complicated by parabolic flow, which distorts and effectively broadens the electrophoretic zones. More detail is available in the cited references on electrophoresis [3-5]. 

I) Zone electrophoresis, an electrophoretic separation technique, conducted In a continuous buffer system. Samples are Injected and move under the applied field they separate into distinguishable zones If their mobilities are sufficiently apart. 

Capillary zone electrophoresis (CZE), micellar capillary electrokinetic chromatography (MECC), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE) are CE modes using continuous electrolyte solution systems. In CZE, the velocity of migration is proportional to the electrophoretic mobilities of the analytes, which depends on their effective charge-to-hydrodynamic radius ratios. CZE appears to be the simplest and, probably, the most commonly employed mode of CE for the separation of amino acids, peptides, and proteins. Nevertheless, the molecular complexity of peptides and proteins and the multifunctional character of amino acids require particular attention in selecting the capillary tube and the composition of the electrolyte solution employed for the separations of these analytes by CZE. 

Many important developments have been made during the last few years but are not yet commercially available. Specifically, there is an active interest in electrospray and continuous flow fast atom bombardment (FAB) for peptides. Applications using supercritical fluid chromatography (SFC) in combination with MB1 and DLI have been slow but continue to attract interest. The current work in capillary zone electrophoresis (with... 

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