Electrophoresis curtain

In preparative continuous free flow electrophoresis, continuous buffer and sample feed are introduced at one end of a thin, rectangular electrophoresis chamber. A schematic is presented in Fig. 11-5. The sample stream is usually introduced through a single port while buffer is introduced through several ports, essentially producing a buffer curtain . Because the buffer streams are introduced independently, it is fairly easy to establish a variety of gradients (e.g., pH, density, ionic strength) across the buffer curtain . 

Free-Flow Electrophoresis. Free-flow electrophoresis is the most common technique for scaling up electrophoresis for commercial application, in this technique, sample compounds are injected into a curtain of buffer which flows between two flat plates, with electrodes parallel to the flow at each end. The electric field is then applied perpendicularly to the flow direction, so that as compounds flow down between the electrodes they separate horizontally and exit the flow field at different locations. 

A main problem in two-dimensional methods is the fate of the buffer salts and this needs careful consideration. The clue to the cause of many erroneous runs and the way to easy and reproducible working conditions are found in a study of the ionic pattern during curtain electrophoresis. 

Fig. 45. Shifts in sodium concentration during two-dimensional electrophoresis with a Veronal-sodium Veronalate buffer, pH 8.6 ionic strength, 0.02 300 volts. There is an increase in sodium from the top toward the bottom of the curtain. There is also a para-anodic zone of sodium increase. B represents the original sodium concentration of the buffer (Pla).

Recently Heremans demonstrated mucopolysaccharides in urine after zone electrophoresis by means of an Alcian blue-acid fuchsin staining technique (HI).3 Applied in our laboratory to a curtain after a run of serum, two fractions, references -)- 86.30 and - - 86.12, were demonstrated but do not seem to be regular components of serum, at least not in that high concentration (Fig. 62c). 

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]. 

Fig. 3.—Distribution of Carbohydrate and Protein Components in a Preparation of Fungal D-Glucose Oxidase in Fractions from Curtain Electrophoresis.6...

This material, described by Nakahara and Fukuoka (Nl), is present only in cancer gastric juice. Upon intraperitoneal injection into mice, it depresses the catalase titer of the liver, kidney, and blood. The relationship of this material to mucopolysaccharides and mucoproteins of gastric juice is not well known. Toxohormone— like material obtained from normal stomach which is biologically inactive, however—was found to have lower polarographic activity than that from stomachs with gastric cancer (M18). Separation of materials with toxohormone activity by continuous electrophoresis on paper curtain and column chromatography is discussed in the following review in this volume. 

Takamura et al. and Wada et gastric juice of normal individuals and patients with histamine-fast anacidity and gastric carcinoma by continuous electrophoresis on paper curtain, and assayed the chemical composition and biological activities of the fractions obtained. Electrophoresis was performed in veronal buffer of pH 8.6 and ionic strength of 0.03, at 225 volts and 7.5-8.25 mA for 24 hours. As in paper strip electrophoresis, 4 major components were obtained, named by the authors (from anode to cathode) Bl, B2, B3, and B4 B3 contained three minor components A, B, and C. In addition, acidic gastric... 

In studies performed in association with J. A. Buckwalter s group from Iowa City, we studied the distribution of ABO(H) blood group substances in the electrophoretic partition of gastric juice (G3a, G6). Several acid and anacid pools of gastric juice were subjected to continuous electrophoresis on paper curtain in borate buffer of pH 9.0, 0.06. The... 

Peeters recently studied the distribution of proteolytic enzymes and proteins on curtain electrophoresis by means of his modified continuous electrophoresis apparatus (Pla) and a new technique which he calls electrochromatography. The viscosity of gastric juice neutralized in situ with phosphate buffer (pH 7.2) was decreased by liquefaction with... 

Ascoxal (a mucolytic drug). Then the gastric juice was concentrated by ultrafiltration and subjected to star electrophoresis in veronal bufiFer of pH 8.6, r/2 0.033. The curtain was stained after oven drying with amido black, azocarmine, or bromophenol blue. Up to 10 protein fractions were localized on the curtain, including pepsin, albumin, and contaminant protease (trypsin ) derived from regurgitated duodenal secretion. Peptic... 

Continuous electrophoresis on paper curtain was applied in our laboratory in fractionating intrinsic factor activity of human gastric juice (G20). In each run, 30-100 mg of dialyzed and lyophilized gastric juice dissolved in borate buffer of pH 9.2 or acetate buffer of pH 4.0 (0.15 and 0.1 ionic... 

G20. Glass, G. B. J., Stephanson, L., Rich, M., and Laughton, R. W., Intrinsic-factor activity of human gastric juice after fractionation by continuous electrophoresis on paper curtain. Brit. J. Haematol. 3, 410-411 (1957). 

K8. Kubo, K., Castro-Curel, Z., Ibanez, N., Glass, G. B. J., and Gode, G. F., Fractionation of gastrone, inhibitory material to gastric secretion by continuous electrophoresis on paper curtain, column chromatography and gel filtration. Gastroenterology 46, 748 (1964). Abstr. 

An ESI interface for the coupling of capillary electrophoresis (CE) and MS was developed by the group of Smith [15-17]. A schematic diagram of the system is shown in Figure 5.4. A hot 2 5-1/min nitrogen curtain gas is used to clean the... 

Electrophoretic separations of solutes are determined primarily by the mass-to-charge ratio of the solute. However, certain electrophoretic separations use gel or paper supports for the electrolyte, and adsorptive and molecular-size fractionations may be as significant as electrophoretic fractionation in these systems. Hall (1970) found that polyacrylamide gel electrophoresis of aquatic humic substances gave size fractionations corresponding to Sepha-dex gel fractionation however, individual fractions were poorly resolved in polyacrylamide gel electrophoresis. Clapp (1957) and Mortensen and Schwendinger (1963) used electrophoresis with electrolyte supporting curtains to separate colorless polysaccharides from humic substances in soil-water extracts. 

In this laboratory, attempts (G6, G8) have been made to purify and crystallize human placental alkaline phosphatase enzyme by a number of procedures involving homogenization with 0.05 M Tris buffer (pH 8.6), extraction with butanol, ammonium sulfate precipitation, exposure to heat, ammonium sulfate fractionation, dialysis, repeated ethanol fractionation, gel filtration with Sephadex G-200 (Fig. 18), continuous curtain electrophoresis on paper (Beckman Model CP), multiple TEAE-cellulose anion exchange chromatography, and equilibrium dialysis. Variant A (electrophoretically fast-moving) of human placental alkaline... 

The world of electromigration separations is sharply divided into two areas. Zone electrophoresis on paper and related procedures have (in spite of their wide applicability to diverse organic compounds) already passed their period of favour. The other branch is represented by the more recent techniques some of which have already became widely accepted (such as isoelectric focusing or separations in polyacrylamide gel) and the others that are at the moment in the centre of a rapid development like displacement electrophoresis (isotachophoresis). This chapter is devoted mainly to analytical procedures such as these which are governing the area of electromigration separations at the moment with a single exception flow deviation (curtain) electrophoresis which will be discussed in more detail because it offers several new dimensions in the separation field. The other preparative procedures are summarized only briefly. 

The two-dimensional separations in which the different driving forces are employed simultaneously are reported here for completeness as, except for curtain electrophoresis, they never reached a wide range of applicability there are (except the already mentioned curtain electrophoresis) electrophoresis with crossed currents employed simultaneously at right angles [24,25], centrifugally accelerated electrophoresis [26] and electromagnetophoresis [27],... 

This procedure represents a preparative version of zone electrophoresis. The apparatus and technique has been previously elaborated [294,295] separation itself is carried out either in a stream of electrolytic solution or on a sheet of cardboard (curtain electrophoresis). In the free flow version the separation is carried out in a cell formed by two parallel glass plates (50 x 50 cm) situated 0.5-1.0 mm apart. It is necessary to ensure an equal and laminar flow of the electrolyte, which is carried out by feeding the buffer through a multichannel peristaltic pump. The sample is continuously applied in the middle near the upper edge of the cuvette (or paper sheet). The electrophoretic separation occurs transversally between vertical electrodes located on the right and left hand side of the separation cuvette (Fig. 6.33). The separated fractions are collected at the lower end of the cell by a system of small communicating vessels or by a multi-channel pump. In the version using paper the... 

The disadvantage of the small scale of FFF separations can be overcome in two-dimensional continuous devices. Here one FFF subtechnique is employed along the flow axis of the channel and another force vector generates a component of motion at right angles to this (cf. free flow or curtain electrophoresis). The result is that different components move along and across the planar accumulation wall, tracing out different trajectories. 

The theory of electrophoresis has been adequately covered in the excellent textbooks of Giddings [1] and Andrews [2] as well as in specific manuals [3], [4]. For discussion on electrophoresis in free liquid media, e.g., curtain, freeflow, endless belt, field-flow-fractionation, particle, and cell electrophoresis the reader is referred to a comprehensive review by Van Oss [5] and to a book largely devoted to continuous-flow electrophoresis [6], Here the focus is mostly on electrophoresis in a capillary support, i.e, in gel-stabilized media, and discussion is limited to protein applications. 

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