Proteomics/Protein Separations- Electrophoresis/Capillary Electrophoresis?
Electrophoresis is the movement or migration of ions or solutes under the influence of an electric field. Capillary electrophoresis is the technique of performing electrophoresis in buffer-filled, narrow-bore capillaries, which normally have internal diameter from 25 to 100 μm.
The instrumentation required for CE is simple( Figure 1) The ends of a capillary are placed in separate buffer reservoirs, each containing an electrode connected to a high-voltage power supply . One of the buffer reservoirs (normally at the anode) is temporarily replaced by sample .The sample is introduced into the capillary via capillary action, pressure, or siphoning. Then an electric potential is applied across the capillary and the separation is performed. Separated analytes can be detected by UV-visible or fluorometric detection directly through the capillary wall near the opposite end (normally near the cathode). Electrophoresis Theory The separation by electrophoresis depends on differences in the migration velocity of ions or solutes through the given medium in the applied electric field. The electrophoretic migration velocity (up) of an analyte toward the electrode of opposite charge is: up = μpE where μp is the electrophoretic mobility and E is the electric field strength.
The electric field strength is a function of the applied voltage divided by the total capillary length. The electrophoretic mobility is directly proportional to the ionic charge of a sample and inversely proportional to any frictional forces present in the buffer. When two species in a sample have different charges or experience different frictional forces, they will separate from one another as they migrate through a buffer solution. The frictional forces experienced by an analyte ion depend on the viscosity (η) of the medium and the size and shape of the ion.
Accordingly, the electrophoretic mobility of an analyte at a given pH is given by:
where z is the net charge of the analyte and r is the Stokes radius of the analyte.
From equation we can see that differences in the charge-to-size ratio of analyte ions cause differences in electrophoretic mobility. Higher charge and smaller size cause greater mobility, whereas
lower charge and larger size cause lower mobility.
Electrophoretic mobility is an indication of fastness of a given ion or solute through a given medium. It expresses the balance of electrical force ( acts in favor of motion) and the frictional force(acts against motion). Since these forces remain in a steady state during electrophoresis electrophoretic mobility is a constant for a given ion under a given set of conditions and therefore is a characteristic property for any given ion or solute. Because of differences in electrophoretic mobility, it is possible to separate mixtures of different ions and solutes by using electrophoresis.
5 Electroosmotic Flow (EOF)
In capillary electrophoresis, the velocity of migration of an analyte will also depend upon the rate of electroosmotic flow (EOF) of the buffer solution.The electroosmotic flow is the bulk flow of liquid through the capillary when current is applied. The capillary tube used for CE is typically an uncoated fused-silica capillary tube and its internal surface has ionisable silanol groups, which readily dissociate giving the capillary wall a negative charge. Therefore, when the capillary is filled with buffer, the negatively charged capillary wall attracts positively charged ions from the buffer solution. This creates electrical double layer and a potential difference called zeta potential close to the capillary wall (figure 2). The electrical double layer includes a rigid layer of adsorbed ions and a diffuse layer. The zeta potential decreases exponentially with increasing distance from the capillary wall surface. When a voltage is applied across the capillary, cations in the diffuse layer are free to migrate towards the cathode, dragging the bulk solution with them. At high pH the silanols are extremely ionized and makes large surface charge of the capillary wall. This increases zeta potential which in turn increases EOF. Thus EOF is very dependent on pH, being large at high pH.
Figure 4: Depiction of the interior of a fused-silica gel capillary in the presence of a buffer solution.
The velocity of the electroosmotic flow, uo can be written as:
uo = μoE where μo is the electroosmotic mobility, which is defined as:
where ζ is the zeta potential of the capillary wall, and ε is the relative permittivity of the buffer solution. The electroosmotic flow of the buffer solution is generally greater than the electrophoretic flow of the analytes and at pH >7 all analytes are carried along with the buffer solution toward the cathode, regardless of their charge. Negatively charged analytes are retained longer in the capilliary due to their conflicting electrophoretic mobilities. Therefore, the observed migration velocity of a solute is related to a combination of both its electrophoretic mobility and the EOF mobility. The velocity (u) of an analyte in an electric field can then be defined as: up + uo = (μp + μo)E
Flow and dispersion
In other separation techniques like HPLC, separations are driven by pressure and that results in frictional forces in places where the mobile phase is in contact with solid surfaces. These frictional forces cause the velocity of mobile phase close to the wall to be zero while that in the center is large resulting in a parabolic flow profile in the capillary(Figure3 ). This profile results in the solute zones being broadened as the move through the capillary, reducing the resolution of the separation. But in CE, since the driving force is EOF, the flow is flat which result much higher resolutions than comparative pressure driven equivalents.
Figure 5: Flow profiles of laminar and electroosmotic flow.
The Electropherogram The data output from CE is an electropherogram, which is a plot of migration time vs. detector response. The detector response is usually concentration dependent, such as UV-visible absorbance or fluorescence. Separated chemical compounds appear as peaks depending on different retention times in an electropherogram. A typical electropherogram shows separation of a three components of mixture of cationic, neutral and anionic solutes.