Preparative ITP Apparatus

Capillary type ITP is useful not only for analytical purposes but also for preparative purposes. Capillary type (CITP) is useful for batch processing of a small amount of sample.

In addition to direct cutting of the capillary section containing the target of interest, preparative methods in CITP can be classified into three types, as shown in Figure 4. Figure 4(A) shows a preparative ITP system reported by Arlinger for the fractionation of the entire sample zones. This system was used in the LKB Tachofrac (Bromma, Sweden, 1983, production discontinued). The zones were swept gradually by a counterflow of a leading electrolyte (ex. 3 iLmin-1) on applying migration current, and the fractions were fixed on a cellulose acetate strip. The separated zones were successively pushed out through a T-branch by applying a counterflow and the zones were continuously fixed on the strip by an electric spray. The linear

Figure 4 Preparative methods in capillary-type isotachophoresis. (A) Arlinger's counterflow method. (B) Modified Arlinger's method. (C) Microsyringe method. (D) Fractionation valve method. A and B, samples; L, leading electrolyte; T, terminating electrolyte; Inj., sample injection port; SP, a counterflow pump; Det., a detector.

Figure 4 Preparative methods in capillary-type isotachophoresis. (A) Arlinger's counterflow method. (B) Modified Arlinger's method. (C) Microsyringe method. (D) Fractionation valve method. A and B, samples; L, leading electrolyte; T, terminating electrolyte; Inj., sample injection port; SP, a counterflow pump; Det., a detector.

velocity of the counterflow was set only a few percent higher than the isotachophoretic migration velocity so as not to dilute the sample by the leading electrolyte. The fractions on the strip can be analysed by immunological and radioactive methods. The zymo-gram technique can be used directly on the strip. The fractions have to be eluted, for analysis by other methods.

A dropwise fractionating method was developed utilizing a counterflow technique. The schematic diagram of the apparatus is shown in Figure 4(B). When the sample zone is pushed out from a T-branch, a spray effect is usually observed due to electrostatic forces. This can be a convenient interfacing technique but it disturbs dropwise fractionation. The electric spray and fluctuation of the drop rate due to electrostatic forces are suppressed by a very simple electrostatic device: As shown in Figure 4(B), the exiting fraction is surrounded by a copper coil, which is connected to a nozzle. The fractions are collected directly into small test tubes on the fraction collector through the coil. By using this technique, complete recovery of the mobile components in the injected samples is possible with minimum risk of loss and contamination. It should be noted, however, that mixing of adjacent sample zones cannot be avoided. The average volume of one drop was ca. 5 |L and the deviation was estimated as +10%. A few nanomoles of the sample components are contained in a drop. The concentration of samples in the fractionated drops or the amount of the target in a fraction was adjustable by changing the flow rate of the leading solution. A typical counterflow of a leading electrolyte was ca. 12 |iLmin-1, which is much higher than the Arlinger-type apparatus.

Figure 4(C) shows another method reported by Kobayashi et al., where the separated sample zones are discontinuously isolated by using a microsyringe. Kobayashi et al. used a potential gradient detector (PGD) with a sample-removal port to fractionate the target zone immediately after the tail of the zone was detected by the PGD. Although the method was not intended for the successive fractionation of the entire sample zones, the ease of operation is notable. This technique was employed for IP-1B and IP-2A instruments (Shimadzu, Kyoto, Japan, production discontinued).

Figure 4(D) shows other discontinuous fractiona-tion technique using a specially designed fractionating valve placed at the end of the separation capillary. After trapping the target zone in the valve, the zone is flushed out.

In addition, the separation tube used was a series of four separation tubes (inner diameter of the tubes, 5-0.5 mm) in order to increase the amount of sample separated. The tube of 5 mm inner diameter was made of acrylic resin and the maximum injectable sample volume was 2.5 mL.

Free-flow apparatus Since no solid media are used in free-flow electrophoresis (FFE), the most important point in instrumentation is the stabilization of the separated zones for any electrophoresis mode. Unstable zones may be caused by unstable operational electrolyte, sample flow, heat convection, density-driven flow, electroosmosis, etc. Bier et al. and Thormann et al. summarized several different methods for stabilizing the zones. A flat-type FFE is treated here, although there are several different approaches using different geometries, such as a thin film between parallel plates, a cylindrical laminar flow between two coaxial cylinders, etc.

Continuous FFE apparatus utilizing a thin flowing fluid was originally designed by Hanig for zone elec-trophoresis. Prusik and Wagner et al. designed and constructed similar apparatus, suggesting that several modes of electrophoresis can be used. At present, the only FFE apparatus available is the Octopus continuous electrophoresis apparatus from Dr. Weber GmbH (Kirchheim-Heimstetten, Germany). By using this apparatus, up to several grams of pure substances can be prepared daily, although the amount depends on the properties of the sample. Figure 5 illustrates the electrolyte circuits of an FFE system (Octopus)

Isotachophoresis Apparatus
Figure 5 Electrolyte circuits of a free-flow electrophoresis apparatus (Octopus, Dr. Weber GmbH). Outer dimensions of separation chamber are 640x180x80 mm, Separation area is 500 x 100 mm (variable thickness 0.4-2.0 mm). P, pumps.

when operated in continuous free-flow isotachophoresis (CFFITP) mode. The effective size of a typical separation chamber is 10-cm wide, 50-cm high and 0.4-mm thick. The sample solution is supplied with a multifold peristaltic pump together with an anolyte and a catholyte. Overflow of the separation chamber is collected as 96 fractions. The flow rate is variable in the range 0.3-100 mL h_1. The sample residence time is variable in the range 1-40 min. High flow rate and small residence time allow stable flow and consequently stable position of zones.

The anolyte and catholyte are circulated by pumps during migration. A dialysis membrane isolates the separation chamber from the electrode compartments. The electrolyte solutions may be denatured (the pH will change) after a few hours operation. The separation chamber can be thermostatted and separation can be monitored with a VIS CCD detection system which can be positioned near the end of the chamber. To obtain pure fractions, the positions of the sample zones at the end of the sample chamber should be stable. The positions are dependent on several factors such as the electric field strength, temperature of the electrolytes, flow rate, and sample and buffer composition. Since these factors are closely correlated with each other, careful control is needed. Sufficient residence time and separation distance are necessary especially when mobility differences are small. For this purpose, a larger separation chamber or a counterflow technique should be used as reported by Prusik (a 50 x 50 cm square chamber with a thickness of 0.5 mm).

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