Moving-boundary apparatus

Figure 8.1 Diagrammatic illustration outlining the physical basis of electrophoretic separation and the moving boundary apparatus employed by Tiselius. (Adapted from Sheehan, Physical Biochemistry, 2000.)...

Several devices are available commercially to measure mobihty. One of these (Zeta-Meter Inc., New York) allows direct microscopic measurement of individual particles. Another allows measurement in more concentrated suspensions (Numinco Instrument Corp., Monroeville, Pa.). The state of the charge can also be measured by a streaming-current detector (Waters Associates, Inc., Framingham, Mass.). For macromolecules, more elaborate devices such as the Tiselius moving-boundary apparatus are used. 

There are a number of complications in the experimental measurement of the electrophoretic mobility of colloidal particles and its interpretation see Section V-6F. TTie experiment itself may involve a moving boundary type of apparatus, direct microscopic observation of the velocity of a particle in an applied field (the zeta-meter), or measurement of the conductivity of a colloidal suspension. 

Douglas et al. [98] have measured protein (serum albumin, ovalbumin, and hemoglobin) mobilities over a range of pH values using a free-flow electrophoresis apparatus and a particle electrophoresis apparatus. They found good agreement between the two measurements however, they also found some differences between their measurements and those reported in the older literature. They attributed the differences to the use of moving-boundary electrophoresis methods in the early experimental work and to differences in... 

The apparatus used to determine the transport number by the moving boundary method is shown in Figure 6.4. It consists of a long vertical tube of uniform cross-section which is fitted with two electrodes at the two ends. Let the electrolyte, the transport number of whose... 

FIG. 12.11 Schematic illustration of a Tiselius-type moving boundary electrophoresis apparatus. 

Experimental Methods.—One of the difficulties experienced in performing transference number measurements by the moving boundary method was the establishment of sharp boundaries recent work, chiefly by Macinnes and his collaborators, has resulted in such improvements of technique as to make this the most accurate method for the determination of transference numbers. Since the earlier types of apparatus... 

An alternative, somewhat simple but less accurate, procedure for measuring transference numbers by the moving boundary principle, utilizes the air-lock method of establishing the boundary. The apparatus for a rising boundary is shown in Fig. 44 the graduated measuring tube A has a bore of about 7 mm., whereas E and F are fine capillaries the top of the latter is closed by rubber tubing with two pinchcocks. The electrodes are placed in the vessels B and C. 

Not only can the Tiselius apparatus be used to indicate the presence of several constituents in a system, but an actual separation of different fractions is sometimes possible. If sufficient time is allowed for the leading boundary to get some distance ahead of the next, the solution between it and the following boundary will consist almost entirely of the faster moving constituent. Since electrophoretic mobilities are, in general, not very different, the boundaries will have moved above the section C or below B in Fig. 131 before there is any appreciable distance between them. If the solution as a whole is given a velocity equal to that of the slower moving boundary but opposite in direction, this boundary will remain stationary at bb in Fig. 131 while the faster one moves steadily ahead, e.g., from 6 into section C. After some time the section C can be... 

By measuring the conductance of several picrates in di-wopropyl ketone at different concentrations, it was shown by the method of Fuoss and Kraus 6 that up to concentration of 01 M there is no detectable triple ion formation. Thus concentrations high enough to satisfy condition (ii) are attainable without the formation of multiple ions. The results of semi-quantitative preliminary experiments indicated that tetraethylammonium and picrate ions had nearly the same mobility in di-wopropyl ketone. This was confirmed by measuring the transport number of the picrate ion by the moving-boimdary method. The conditions for the successful use of the moving-boundary method have been fully examined by Longsworth and Maclnnes.7 A simplified apparatus was used and is shown in fig. 3 camphor-sulphonate was found to be a suitable indicator ion. 

Development of the technique was slow until the early 1960s, when suitable apparatus became commercially available. Several names have been used for this separation technique ion migration method, moving boundary method, displacement electrophoresis, and cons electrophoresis. The term used here, isotachophoresis (ITP), is based on the important associated phenomenon of the identical velocities of the sample zones in the steady state. 

A complete apparatus for moving boundary measurements is shown in Fig. 7. It includes two electrode vessels A and A the graduated tube D and two sets of disks, 5, C and B C. All the disks are similar to the one shown in Fig. 6c. The upper pair of disks is used for descending boundaries and the lower pair for rising boundaries. 

Figure 11.11 (a) A moving boundary experiment showing a schematic apparatus, (b) Movement of the boundary with time. 

Moving Boundary Electrophoresis An indirect electrophoresis technique for particles too small to be viewed. This principle is used in the Tiselius apparatus. Here a colloidal dispersion is placed in the bottom of a U-tube, the upper arms of which are filled with a less dense liquid that both provides the boundaries and makes the connections to the electrodes. Under an applied electric field the motions of the ascending and descending boundaries are measured. 

Tiselius Apparatus An apparatus for the determination of electrophoretic mobilities. See Moving Boundary Electrophoresis. 

In comparison to other electrophoretic techniques moving boundary electrophoresis has only Umited applications and can be used for testing, among other things, purity of substances. Its advantage is based on the fact that rather simple equipment can be used (in comparison with isotachophoretic apparatus) zone boundaries are detected with a conductivity and a photometric detector and the whole separation is carried in narrow bore tubes. 

Figure 7.3.4 Tiselius moving boundary electrophoresis apparatus. [After Shaw, D.J. 1980. Introduction to Colloid and Surface Chemistry, 3rd edn. London Butterworths. With permission.]...

The moving-boundary method for the measurement of transference numbers has been brought to a high state of perfection. A schematic diagram of the apparatus is shown in Fig. 31.6. A tube has two electrodes fixed at the ends and contains two solutions having an... 

Martin and Everaerts published a paper in 1967 which gave a new impulse. Their capillary equipment for displacement electrophoresis had already the character of the present devices. They used as a detector a thermocouple glued on the outside wall of a thin-walled separation capillary. The principle of and the equipment for the moving boundary analysis with a detection performed potentio-metrically was reported by Hello in 1968. One year later, Fredriksson developed the analytical apparatus for the displacement electrophoresis . He used a conductivity detector, successfully separated low-molecular fatty acids and performed quantitative an dyses by measuring the lengths of zones. 

It was not until the work of Tiselius in the 1930s that the potential of electrophoresis as a biochemical tool was realized. Tiselius developed the use of electrophoresis for separating proteins in suspension on the basis of their charge. He optimized the geometry and temperature of the system in an apparatus known as the Tiselius moving boundary system. Detection of the separated components was carried out by detecting concentration changes in optical refraction. 

Macinnes DA, Cowperthwaite lA, Blanchard KC (1926) The moving-boundary method for determining transference numbers. V. A constant current apparatus. J Am Chem Soc 48 1909-1912... 

Figure 12.5 Moving Boundary Electrophoresis (A) Schematic diagram of separation of protein mixture (x.y.z) by moving boundary eletrophoresis (i) shows the initial position after the apparatus has been filled with protein and buffer solutions. (W shows the flnal position after separation. All proteins have negative charge at the pH of the buffer, x has the highest mobility and z has the lowest mobility. Note that only some amount of x and z have separated completely. It has not been possible to separate y. (Diffusion has not been taken into account in this figure for the sake of simplicity).

The apparatus for moving boundary electrophoresis itself and the associated Schlieren optics are very costly. This is another restriction for the use of free electrophoresis. In comparison, the apparatus for zone electrophoresis is simple and cheaper. 

Fig. 7. Apparatus used by Burton for the determination of the electrophoretic velocity by the moving boundary method.

---