Capillary Electrophoresis - Modes of Separation

Modes of Separation

The separation of compounds by capillary electrophoresis is dependent on the differential migration of analytes in an applied electric field. The electrophoretic migration velocity of an analyte toward the electrode of opposite charge is:

where is the electrophoretic mobility and E is the electric field strength. The electrophoretic mobility is 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 is the net charge of the analyte and is the Stokes radius of the analyte. The Stokes radius is given by:

where is the Boltzmann constant, and is the temperature, D is the diffusion coefficient. These equations indicate that the electrophoretic mobility of the analyte is proportional to the charge of the analyte and inversely proportional to its radius. The electrophoretic mobility can be determined experimentally from the migration time and the field strength:

where is the distance from the inlet to the detection point, is the time required for the analyte to reach the detection point (migration time), is the applied voltage (field strength), and is the total length of the capillary. Since only charged ions are affected by the electric field, neutral analytes are poorly separated by capillary electrophoresis.

The velocity of migration of an analyte in capillary electrophoresis will also depend upon the rate of electroosmotic flow (EOF) of the buffer solution. In a typical system, the electroosmotic flow is directed toward the negatively charged cathode so that the buffer flows through the capillary from the source vial to the destination vial. Separated by differing electrophoretic mobilities, analytes migrate toward the electrode of opposite charge. As a result, negatively charged analytes are attracted to the positively charged anode, counter to the EOF, while positively charged analytes are attracted to the cathode, in agreement with the EOF as depicted in figure 3.

The velocity of the electroosmotic flow, can be written as:

where is the electroosmotic mobility, which is defined as:

where is the zeta potential of the capillary wall, and is the relative permittivity of the buffer solution. Experimentally, the electroosmotic mobility can be determined by measuring the retention time of a neutral analyte. The velocity of an analyte in an electric field can then be defined as:

Since the electroosmotic flow of the buffer solution is generally greater than that of the electrophoretic flow of the analytes, all analytes are carried along with the buffer solution toward the cathode. Even small, triply charged anions can be redirected to the cathode by the relatively powerful EOF of the buffer solution. Negatively charged analytes are retained longer in the capillary due to their conflicting electrophoretic mobilities. The order of migration seen by the detector is shown in figure 3: small multiply charged cations migrate quickly and small multiply charged anions are retained strongly.

Electroosmotic flow is observed when an electric field is applied to a solution in a capillary that has fixed charges on its interior wall. Charge is accumulated on the inner surface of a capillary when a buffer solution is placed inside the capillary. In a fused-silica capillary, silanol (Si-OH) groups attached to the interior wall of the capillary are ionized to negatively charged silanoate (Si-O-) groups at pH values greater than three. The ionization of the capillary wall can be enhanced by first running a basic solution, such as NaOH or KOH through the capillary prior to introducing the buffer solution. Attracted to the negatively charged silanoate groups, the positively charged cations of the buffer solution will form two inner layers of cations (called the diffuse double layer or the electrical double layer) on the capillary wall as shown in figure 4. The first layer is referred to as the fixed layer because it is held tightly to the silanoate groups. The outer layer, called the mobile layer, is farther from the silanoate groups. The mobile cation layer is pulled in the direction of the negatively charged cathode when an electric field is applied. Since these cations are solvated, the bulk buffer solution migrates with the mobile layer, causing the electroosmotic flow of the buffer solution. Other capillaries including Teflon capillaries also exhibit electroosmotic flow. The EOF of these capillaries is probably the result of adsorption of the electrically charged ions of the buffer onto the capillary walls. The rate of EOF is dependent on the field strength and the charge density of the capillary wall. The wall's charge density is proportional to the pH of the buffer solution. The electroosmotic flow will increase with pH until all of the available silanols lining the wall of the capillary are fully ionized.