Tiselius apparatus separations

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... 

Fig. 2. pH Activity curves of two anodal-moving components separated from concentrated human gastric juice in the Tiselius apparatus. , Major, faster component, mobility 2.99 X 10— cm /volt/sec o, minor, slower component. Electrophoresis was carried out in 33 mM acetate buffer, pH 2.5. Protein concentration, 0.51 g/100 ml. Human serum albumin as substrate temp. 37° time 3 hr. From Taylor (T15). 

Arne Tiselius announced his electrophoresis apparatus, which was enormous both in size and expense, in 1937. Electrophoresis was designed for the separation and investigation of proteins, and the technique has had its greatest impact in biology and biochemistry.174 The development of electrophoretic methods after Tiselius has been... 

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.)...

This technique, although described in the literature, has been given little attention. Isoelectric focusing without stabilizing media can be done in an apparatus similar to Tiselius free zone electrophoresis [152]. Separation itself occurs in a horizontal quartz tube that is rotated at 40 rpm to counteract convective forces [153]. The pH gradient is evolved without the addition of ampholytes, however this method leads to either too steep or too shallow gradients and is therefore not practically applicable. Another alternative method for free solution isoelectric focusing is the application of polyethylene coils however this procedure makes use of ampholines [154]. 

Tiselius (15) described the focusing of ampholytes at their respective isoelectric points as a dynamic equilibrium between electrophoretic migration and diffusion. He used the method on a mixture of egg albumin and hemoglobin. Separation was poorer than by electrophoresis alone. Synge (16) and Sanger and Tuppy (17) used isoelectric focusing for a rough separation of amino acids. This apparatus had four chambers. 

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. 

It is possible with this apparatus to separate rather large amounts of substances if many sheets of paper are used on top of each other. With six sheets Kunkel and Tiselius (1951) were able to separate the proteins in 1 ml. of serum. 

Tiselius (1941) applied the principle of isoelectric fractionation by electrical transport to proteins using the apparatus shown in Fig. 9. It was a 12-compartment perspex apparatus with parchment paper as cathodic, leather as anodic, and cotton flannel as intermediate membranes. Stirring was effected by horizontally oscillating glass rods. After the stationary state was reached, all compartments were emptied instantaneously and dmultaneously. A specird arrangement for that purpose was described. In Table I can be seen the separation which was effected between egg albumin and hemoglobin in 0.005 N sodium sulfate solution after a 24 hours run. It is seen that the separation was almost complete, only one cell containing both proteins. 

The separation is carried out at the density maximum of water. Tiselius showed by a theoretical discussion and experiments that the simple arrangement of cooling the apparatus down to - -4°C. increased the maximum possible load of the apparatus, and thus the separation speed, quite considerably. 

---