Capillary electrophoresis (CE) has developed into an extremely powerful analytical technique in recent years (Heiger, 1992). Along with advances in instrumentation and separation methodologies, a wide range of applications has been developed in many areas including chemical, biotechnical, pharmaceutical, and environmental analysis (Li, 1992).

Electrophoresis is the differential movement of ions by attraction or repulsion in an electric field. In high-performance capillary electrophoresis (HPCE), separation is performed in narrow-bore capillaries, typically of 25-75 micrometers (mm) inner diameter (id). In a CE system, the ends of the capillary are connected to electrodes, which are connected to a high voltage power supply. The capillary ends are placed into buffer reservoirs, and the capillary is filled with a buffer identical to that in the reservoirs. The sample is introduced into the capillary by replacing one of the buffer reservoirs with a sample reservoir (usually at the anode end); the sample may be injected either electrokinetically or hydraulically. After the buffer reservoir is replaced, the electric field is applied and the separation is performed. Either on-line or off-line optical detection can be made at the cathode end of the capillary.

Separation by electrophoresis is based on differences in solute velocities in an electric field. The velocity of an ion can be given by the following equation:

(1.1)

where: v = ion velocity

m_e = electrophoretic mobility

E = applied electric field

The electric field is a function of the applied voltage and the capillary length. The mobility for a given ion and medium is a constant which is characteristic of that ion, and may be defined as follows:

(1.2)

where; q = ion charge

h = solution viscosity

r = ion radius

A fundamental part of HPCE operation is the electroosmotic flow (EOF). EOF is the bulk flow of liquid in the capillary. Most capillaries used for CE today are made of fused-silica, which contains surface silanol groups (Li, 1992; Heiger, 1992).

The interface between the fused-silica tube wall and the electrophoretic buffer consists of three layers; the negatively charged silica, the immobile layer, and the diffuse layer of cations adjacent to the surface of the silica, which tend to migrate toward the cathode. The migration of cations results in an accompanying migration of fluids through the capillary; this migration causes the EOF.

The EOF is significantly greater than the electrophoretic mobility of the individual ions contained in the sample. Consequently, both anions and cations can be separated in the same run. Cations are attracted toward the cathode and their speed is augmented by the EOF. Although anions are electrophoretically attracted toward the anode, they are carried toward the cathode with the electroosmotic flow of the buffer. Cations with the highest charge/mass ratios will migrate first, followed by the cations with reduced ratios. Next, neutral components migrate with the same velocity as the EOF, and finally, the anions migrate. Those anions with lower charge/mass ratios migrate faster than those with greater ones.

A unique feature of the EOF in the capillary is the flat flow profile. Since the driving force of the flow is uniformly distributed along the capillary wall, there is no pressure drop within the capillary and flow is nearly uniform throughout.

This increases the resolution in separations by reducing the band broadening of the analyte peak during its passage along the capillary (Heiger, 1992).

Electroosmotic flow is important in CE and must be controlled. For example, at a high pH the EOF may be too rapid. This may result in the elution of the solute before separation has taken place. Conversely, at a low pH the negatively charged wall can cause adsorption of cationic solutes through coulombic interactions (Heiger, 1992). Successful separations are usually obtained when the conditions optimize both EOF and solute mobility properties. Table 1 dtails several methods that may be used to established optimal conditions.

The analytical parameters for capillary electrophoresis can be described in similar terms as those for column chromatography. These parameters include migration time, mobility, and dispersion. The migration time of a solute refers to the time required for it to migrate from the point of injection to the point of detection. Migration time, along with other experimental parameters may be used to calculate the apparent mobility, given by the following equation:

(1.3)

where; m_a = m_e + m_EOF

V = applied voltage

l = effective capillary length

L = total capillary length

t = migration time

E = electric field

In the presence of the EOF, the measured mobility is called the apparent mobility.

The effective mobility can be determined from the apparent mobility by independently measuring the EOF with a neutral marker that moves through the capillary at a velocity equal to the EOF (Terabe, 1990; Li, 1992).

Dispersion is the spreading of the solute zone, which results from differences in solute velocity within the zone, and can be defined as the baseline peak width, w_b;

(1.4)

where: s = standard deviation of peak width (in time length or volume)

and the separation efficiency, expressed in the number of theoretical plates, N, can be obtained by using the following equation:

(1.5)

where: D = diffusion coefficient (found in tables)

The theoretical plate number can also be determined from the electropherogram by using the following equation:

(1.6)

where: t = migration time

w_1/2 = temporal peak width at ½ height

In practice, the measured efficiency is usually lower than the calculated efficiency because the theoretical calculation only accounts for zone broadening due to longitudinal diffusion (Heiger, 1992).

There are a number of other factors that contribute to zone broadening, including Joule heating and the adsorption of samples to the capillary wall. Joule heating is a result of the heat generated by the passage of an electrical current through the capillary.

The temperature increase depends on the power generated, and is determined by the capillary dimensions, the conductivity of the buffer, and the applied voltage. Joule heating causes a temperature gradient within the capillary; significantly elevated temperatures will result when the power generation exceeds the dissipation. Table 2 provides methods to control Joule heating and temperature gradients in the capillary.

Table 1 Methods to Control Electroosmotic Flow

Source: David N. Heiger, High Performance Capillary Electrophoresis: An Introduction, (France: Hewlett Parkard Company, 1992)21.

Table 2 Methods to Control Joule Heating and Temperature Gradients

Source: David N. Heiger, High Performance Capillary Electrophoresis: An Introduction, (France: Hewlett Packard Company, 1992) 30.

Interaction between the solute and the capillary wall is detrimental in HPCE. The primary causes of adsorption to the fused-silica walls are ionic interactions between cationic solutes and the negatively charged wall, and hydrophobic interactions. The large surface area-to-volume ratio of the capillary, which is beneficial for heat transfer, actually increases the likelihood of adsorption. There are several strategies employed to reduce the solute-wall interaction. For example, an increase in the concentration of the buffer will result in a decrease in the solute interaction by reducing the effective surface charge. Another approach is coating the capillary wall. Coating the wall causes a decrease in the solute adsorption by decreasing the free energy of interaction. Coatings can take various forms, including buffer additives and covalent modification of the capillary wall (Heiger, 1992). There are several different modes of capillary electrophoresis. The following table presents each mode, and a brief description of its separation mechanism.

Table 3 Different Modes of High Performance Capillary Electrophoresis

Source: S. F. Y. Li, Capillary Electrophoresis: Principles, Practice and Applications, (Amsterdam: Elseiver, 1992) 4-12.

The CE method used in this experiment micellar electrokinetic chromatography (MEKC). In MEKC, the main separation is based on solute partitioning between the micellar phase and the solution phase. Micelles form in solution when a surfactant is added to water in a concentration above its critical micelle concentration (cmc). Micelles consist of aggregates of surfactant molecules with typical lifetimes of less than 10 ms (Li, 1992). The most commonly used surfactant in MEKC is sodium dodecyl sulphate (SDS).

According to Li (1992), SDS micelles can be considered as small droplets of oil with a highly polar surface which is negatively charged. Even though the micelles are negatively charged, they migrate toward the cathode end of the capillary because of the EOF. However, the micelles travel at a rate slower than that of the bulk aqueous flow because of their attraction toward the anode. Neutral molecules partition in and out of the micelles based on their hydrophobicity. For example, a very hydrophillic, neutral molecule like methanol will spend almost no time inside the micelle and will essentially migrate at the same rate as the bulk aqueous flow. Conversely, an extremely hydrophobic neutral molecule such as sudan III will spend nearly all the time inside the micelle and will be eluted with the micelle (Terabe, 1990).

In some cases, modifiers are added to the electrophoretic solution to enhance the separation efficiency. One of the commonly used modifiers is cyclodextrin. Cyclodextrin (CD) is an electrically neutral, organic polymer, and its outside surface is hydrophillic; it does not interact with the micelle. Therefore, CD in the micellar solution exists as another phase, which migrates with a velocity identical to that of the bulk aqueous solution and is capable of selectively solubilizing certain solutes depending on their size, shape, and hydrophobicity. When a highly hydrophobic substance, which is insoluble in water, is injected into the CD-MEKC system, it will distribute itself between the micelle and the CD cavity (Li, 1992).

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Li, S. F. Y. Capillary Electrophoresis: Principles, Practice and Applications. Journal of Chromatography Library. 52. Amsterdam: Elsevier, 1992.

Nakagawa, T. "Division of Colloid Surface Chemistry Newsletter"., Chemical Society of Japan 6 (1981): No. 3, p.1.

Ong, C. P., et al. "Retention of Eleven Priority Phenols Using Micellar Electrokinetic Chromatography." Journal of Chromatography 516 (1990): 263-270.

Ong, C. P., H. K. Lee, and S. F. Y. Li. "Separation of Phthalates by Micellar Electrokinetic Chromatography." Journal of Chromatography 542 (1991): 473-481.

Rasmussen, Henrik T., Lisa K. Gobel, and Harold M. McNair. "Micellar Electrokinetic Chromatography Employing Sodium Alkyl Sulfates and Brij 35." Journal of Chromatography 517 (1990): 549-555.

Terabe, Shigeru, et al. "Separation of Highly Hydrophobic Compounds by Cyclodextrin- Modified Micellar Electrokinetic Chromatography." Journal of Chromatography 516 (1990): 23-31.

-----. "Electrokinetic Separations with Solutions and Open-tubular Capillaries." Analytical Chemistry 56 (1984): 111-113.

Yan, Chao, et al. "Capillary Electrochromatography: Analysis of Polycyclic Aromatic Hydrocarbons." Analytical Chemistry 67 (1995): 2026-2029.

Yik, Yin Fun, et al. "Separation of Selected PAH’s by Using High Performance Capillary Electrophoresis with Modifiers." Environmental Monitoring and Assessment 19 (1991): 73-81.

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