Moving Boundary Electrophoresis

 

The moving boundary method was the first to be used by Tiselius to demonstrate the efficacy of the electrophortic process. The apparatus consisted of a U tube the horizontal lower portion of the U tube being filled with a mixture of the substances under examination dispersed in a suitable buffer. The two vertical limbs were filled solely with buffer and the cathode and anode dipped into the buffer at the top of each limb respectively. The system is depicted in figure 4.

 

Figure 4. Moving Boundary Electrophoresis

 

Assume that the sample contains cations A⁺, B⁺, C⁺ and anions A^-, B^- and C^-. The cations and anions will be attracted to the cathode and anode respectively and bands of ions will be formed in both limbs. In the initial stages, in the limb containing the cathode the highest band will consist exclusively of the fastest moving cation (A⁺) (the cation with the greatest mobility). The next band will contain both (A⁺) the cation with the greatest mobility and (B⁺) the cation with the next greatest mobility. While the third, and lowest band, will contain a mixture of all three cations including the cation with the lowest mobility. In the limb containing the anode exactly the same process will take place the highest band will consist exclusively of the fastest moving anion (A^-) (the anion with the greatest mobility). The next band will contain both (A^-) the anion with the greatest mobility and (B^-) the anion with the next greatest mobility. While the third, and lowest band, will contain a mixture of all three anions including the anion with the slowest mobility.

 

The bands are best identified and measured using the Schleren optical system. The Schleren optical arrangement consists of a collimated light source directed on, or from behind, the object and any changes in the refractive index resulting from changes in density distort the beam. This results in a variation in light intensity that can be visualized directly with a shadowgraph technique. In the Schleren optical system a knife-edge is placed at the focal point of the collimating lens that blocks out half the light. If the flow has uniform density this will merely reduce intensity of the light activating the photograph. However, if the density varies then a series of light and dark patches will be produced corresponding to positive and negative density gradients. If the knife-edge is used, which defines the Schleren system, then the output will be proportional to the first derivative or density gradient along the tube. If the knife-edge is not employed then output of light is proportional to the second derivative of the density and the result is called a shadowgraph. A typical density length curve from a moving boundary separation is shown in figure 5

 

 

Figure 5. A Separation Demonstrating the Moving Boundary Technique

 

It is seen that the moving boundary trace closely resembles a separation that would be obtained chromatographically by frontal analysis. In an exactly analogous manner only the first step contains a single substance and as more steps are produced so they become more contaminated with following electrolytes. However, if the separation was monitored was monitored employing the Schleren optical system it would produce a curve relating the first differential to distance travelled (similarly to the derivative of a frontal analysis curve. An example is given figure 6.

 

 

 

Figure 6. The Separation of Some Blood Components Presented as a Curve Relating the First Derivative of the Density Against Distance Travelled