Continuous-flow paper electrophoresis

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. 

Figure 16.2 Diagram of the apparatus used for continuous-flow paper electrophoresis.

The apparatus used for continuous-flow paper electrophoresis can be used for separation with an elution rather than a continuous-flow method. The sample is spotted at the top of the paper and only the buffer solution is continuously added. If the electrophoresis is stopped before the sample components drip from the bottom of the paper, the technique combines the separating ability of descending paper chromatography (separation occurring vertically) with that of electrophoresis (separation occurring horizontally) to yield spots simultaneously developed in two dimensions. 

Some coupled systems allow measurement of the main N and P forms (nitrate, ammonia and orthophosphates) [22,27,29], among which is a system based on membrane technology in combination with semi-micro continuous-flow analysis (pCFA) with classical colorimetry. With the same principle (classical colorimetry), another system [30] proposes the measurement of phosphate, iron and sulphate by flow-injection analysis (FIA). These systems are derived from laboratory procedures, as in a recent work [31] where capillary electrophoresis (CE) was used for the separation of inorganic and organic ions from waters in a pulp and paper process. Chloride, thiosulphate, sulphate, oxalate,... 

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]. 

Figure 7.3. In continuous deflection electrophoresis, flow down a paper sheet or between closely spaced plates carries different solutes, which are gradually separated by electrophoresis along axis x, to different collection ports. However, the flow is classified as passive because it does not enhance the separation occurring along the electrical-field axis. The role of flow is to aid continuous sample collection, not to fundamentally alter the separation process.

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... 

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