The Process Of Electrophoretic Separation 341 Electrophoretlc Separation

The velocity of migration of a charged analyte in an electric field depends on its electrophoretic mobility and on the magnitude of the applied electric field.

to the detector and illuminates a section of the capillary. Fluorescence is collected by the ellipsoidal mirror and focused back onto the pbotomulliplier tube. To reduce unwanted laser light, a centered hole in the mirror allows most of the beam to pass. A beam block is used to attenuate scattered laser light. (Reprinted with permission from Schwartz and Guttman, Beckman ¬°instruments Primer Series, Vol. VII, 1995. Copyright: Beckman Instruments, Inc.)

to the detector and illuminates a section of the capillary. Fluorescence is collected by the ellipsoidal mirror and focused back onto the pbotomulliplier tube. To reduce unwanted laser light, a centered hole in the mirror allows most of the beam to pass. A beam block is used to attenuate scattered laser light. (Reprinted with permission from Schwartz and Guttman, Beckman ¬°instruments Primer Series, Vol. VII, 1995. Copyright: Beckman Instruments, Inc.)

Electrophoretic mobility in turn increases with magnitude of the analyte's charge and decreases with its radius. That is, if you have two analytes of the same size, the one with the greatercharge will move faster. But if you have two analytes of different size and the same charge, the smaller one will move faster.

3.4.2 Electroosmosls

Besides electrophoretic migration, analytes in CE move by a process called electroosmosis (or electroendoosmosis). This phenomenon, occurring also in slab gel electrophoresis, produces electroosmotic flow, the electrically driven flow of the liquid within the capillary. However, while in slab gel electrophoresis the gel matrix reduces EOF to an annoyance, in CE this liquid flow can have a significant effect on the separation process.

ElectroosmoKis originates from the charges present on the inner surface of the capillary. In the case of fused silica capillaries, the origin of these charges is the dissociation of the silanol (StO") groups of the glass. This dissociation occurs at pH values higher than 2. These negative charges on the wall attract positively chargcd cations (e.g., Na; K in the buffer) to neutralize them, creating an ionic double layer and, consequently, a potential difference, the zeta potential. This situation obtains before the electrophoresis process begins.

When electrophoresis begins, the voltage difference set up in the capillary causes the migration of the mobile positive ions (cations) toward the cathode. This ionic movement in turn osmotically drags fluid, the water in the capillary, in the same direction. It is this movement of fluid that generates the EOF. The velocity of this flow is increased with the dielectric constant of the fluid and the magnitude of the zeta potential, and decreased by the solution's viscosity.

With bare (uncoated) silica capillaries, osmotic flow is from the anodic to the cathodic end. However, if the capillary is coated with a positive surface, the osmotic flow would be reversed. Although extremely variable in dependence of experimental conditions, EOF is generally in the order of fractions of milliliters per minute; it can be empirically measured by injecting a neutral marker (e.g., acetone).

A peculiarity of EOF is that originating at the walls of the capillary, its flow profile is almost flat, without the parabolic shape typical of pressure-generated laminar flow, as in HPLC (Fig. 3.3). It is of interest to note that in comparison to a parabolic profile, a flat profile limits the broadening of the zones during migration in the capillaries.

The usual arrangement of a capillary electropherograph necessitates that injection take place at the anodic end, with detection occurring close to the cathodic end of the capillary. Considering also that EOF is generally oriented toward the cathode and that it is greater than the electrophoretic velocity of most analytes, it follows that cationic, neutral, and anionic analytes will reach the detector in that order.

The actual migration velocity of cations and anions will result from the algebraic summation of the electrophoretic mobilities of the individual ionic electroosmotic flow profile

-y pressure driven flow profile

Figure 3.3 Schematic representation of the flow profiles of EOF and pressure-generated laminar flow.

Figure 3.3 Schematic representation of the flow profiles of EOF and pressure-generated laminar flow.

species and that of EOF. All neutral species migrate at the velocity of the EOF, in a single zone (peak), and consequently, cannot be resolved from one another.

In the presence of the EOF, the migration velocity of the analytes will increase with the mobilities of EOF, the analyte, and the voltage and will decrease with capillary length.

EOF can be effectively controlled by changing several experimental conditions. For example, an increase in buffer pH produces an increase in EOF, as long as it increases the degree of dissociation of the wall silanols. Also the ionic strength of the buffer influences the zeta potential and, consequently, the EOF as follows: the less the ionic strength, the less the neutralization of wall charges and therefore the greater will be the zeta potential and the EOF. The addition of organic solvents to the buffer affects both the zeta potential and buffer viscosity. Also, increasing the applied voltage increases the Joule heating of the buffer, with consequent reduction in viscosity and increase of EOF. Buffer additives (e.g., surfactants, methylcellulose, polyacrylamide, quaternary amines) can change quantitatively and/or qualitatively the wall charges, thus reducing or, if cationic, even reversing EOF. Similarly, capillary wall coatings (e.g., polyacrylamide, proteins, amino acids, PEG, PVA, C8, C18) that covalently bond to the surface (often by silylation), and, in some cases, those that are only physically adsorbed (e.g., cellulose) can effect the EOF.

Was this article helpful?

0 0

Post a comment