Electrophoresis anode

The basic instrumentation for capillary electrophoresis is shown in Figure 12.41 and includes a power supply for applying the electric field, anode and cathode compartments containing reservoirs of the buffer solution, a sample vial containing the sample, the capillary tube, and a detector. Each part of the instrument receives further consideration in this section. 

Capillary Zone Electrophoresis The simplest form of capillary electrophoresis is capillary zone electrophoresis (CZE). In CZE the capillary tube is filled with a buffer solution and, after loading the sample, the ends of the capillary tube are placed in reservoirs containing additional buffer solution. Under normal conditions, the end of the capillary containing the sample is the anode, and solutes migrate toward... 

Capillary zone electrophoresis also can be accomplished without an electroosmotic flow by coating the capillary s walls with a nonionic reagent. In the absence of electroosmotic flow only cations migrate from the anode to the cathode. Anions elute into the source reservoir while neutral species remain stationary. 

The other reactions at the electrodes produce acid (anode) and base (cathode) so that there is a possibiUty of a pH gradient throughout the electrophoresis medium unless the system is well buffered (see Hydrogen-ion activity). Buffering must take the current load into account because the electrolysis reactions proceed at the rate of the current. Electrophoresis systems sometimes mix and recirculate the buffers from the individual electrode reservoirs to equalize the pH. 

Electrophoresis of water solutions of these salts and using steel as either the anode or the cathode, respectively, liberates the water-insoluble polymer coating through the reaction ... 

Figure 1. Scale drawing of glass plates. BSA was adsorbed into the left position (2.5 cm long) of the plates, which subsequently was the cathodal end. After electrophoresis the plates were cut up into 4 equal pieces, each 3.5 cm long (numbered 1 to 4) for Y-counting plus an anodal end (H), used for handling, which remained uncounted. Reproduced from Absolom et al. (8) with permission from Verlag Chemie Gmb H.

Figure 7.1 Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing agents, which disrupt disulfide linkages). The electric field is applied across the gel after the protein samples to be analysed are loaded into the gel wells. The rate of protein migration towards the anode is dependent upon protein size. After electrophoresis is complete, individual protein bands may be visualized by staining with a protein-binding dye (a). If one well is loaded with a mixture of proteins, each of known molecular mass, a standard curve relating distance migrated to molecular mass can be constructed (b). This allows estimation of the molecular mass of the purified protein...

Isotachophoresis. In isotachophoresis (ITP), or displacement electrophoresis or multizonal electrophoresis, the sample is inserted between two different buffers (electrolytes) without electroosmotic flow. The electrolytes are chosen so that one (the leading electrolyte) has a higher mobility and the other (the trailing electrolyte) has a lower mobility than the sample ions. An electric field is applied and the ions start to migrate towards the anode (anions) or cathode (cations). The ions separate into zones (bands) determined by their mobilities, after which each band migrates at a steady-state velocity and steady-state stacking of bands is achieved. Note that in ITP, unlike ZE, there is no electroosmotic flow and cations and anions cannot be separated simultaneously. Reference 26 provides a recent example of capillary isotachophoresis/zone electrophoresis coupled with nanoflow ESI-MS. 

In practice isotachophoresis is usually performed in narrow tubes with electrodes at either end and is one form of capillary electrophoresis. For the separation of a particular type of ion, e.g. an anion, two buffered electrolyte solutions are selected that have different anions but a common cation with a buffering capacity. One of the anions (termed the leading electrolyte) should show a greater mobility than the other anion and occupies the anodic end of... 

Figure 7.6 Rocket electrophoresis. At pH 8.6 most proteins move towards the anode and precipitation occurs where the antigen and the antibody (in the gel) are in equivalent proportions. The size of the resulting rocket -shaped pattern of precipitate is proportional to the original concentration of antigen. The immunoglobulins show very little electrophoretic mobility at pH 8.6 and so remain in the gel during the process.

Figure 8.1 Exploded view of an electrophoresis cell. The components of the Bio-Rad Mini-PROTEAN 3 are shown. The inner chamber can hold one or two gels. It contains an electrode assembly and a clamping frame. The interior of the inner assembly constitutes the upper buffer compartment (usually the cathode compartment). The chamber is placed in the tank to which buffer is added. This constitutes the lower (anode) buffer compartment. Electrical contact is made through the lid.

Proteins are positively charged in solutions at pH values below their pi and negatively charged above their pis. Thus, at pH values below the pi of a particular protein, it will migrate toward the cathode during electrophoresis. At pH values above its pi, a protein will move toward the anode. A protein at its pi will not move in an electric held. 

Separation is performed using free-zone electrophoresis, where the capillary is filled with a separating buffer at a defined pH and molarity. This buffer is also called a BGE. During separation, the polarity is set to cathodic or anodic mode, also called normal and reverse mode, depending on the charge of the molecule cation or anion. For anions, the capillary is usually dynamically coated with an electroosmotic flow (EOF) modifier to reverse the EOF and separate the analytes in the co-electroosmotic mode. 

Isoelectric points are useful concepts for the separation and purification of amino acids and proteins using electrophoresis. Under the influence of an electric field, compounds migrate according to their overall charge. As we have just seen for amino acids, this very much depends upon the pH of the solution. At the isoelectric point, there will be no net charge, and, therefore, no migration towards either anode or cathode. 

According to Equation 6.6, the velocity of the EOF is directly proportional to the intensity of the applied electric held. However, in practice, nonlinear dependence of the EOF on the applied electric held is obtained as a result of Joule heat production, which causes the increase of the electrolyte temperature with consequent decrease of viscosity and variation of all other temperature-dependent parameters (protonic equilibrium, ion distribution in the double layer, etc.). The EOF can also be altered during a run by variations of the protonic concentration in the anodic and cathodic electrolyte solutions as a result of electrophoresis. This effect can be minimized by using electrolyte... 

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