30.3: Applications of Capillary Electrophoresis

There are several different forms of capillary electrophoresis, each of which has its particular advantages. Several of these methods are described briefly in this section.

Capillary Zone Electrophoresis (CZE)

The simplest form of capillary electrophoresis is capillary zone electrophoresis. In CZE we fill the capillary tube with a buffer and, after loading the sample, place the ends of the capillary tube in reservoirs that contain additional buffer. Usually the end of the capillary containing the sample is the anode and solutes migrate toward the cathode at a velocity determined by their respective electrophoretic mobilities and the electroosmotic flow. Cations elute first, with smaller, more highly charged cations eluting before larger cations with smaller charges. Neutral species elute as a single band. Anions are the last species to elute, with smaller, more negatively charged anions being the last to elute.

We can reverse the direction of electroosmotic flow by adding an alkylammonium salt to the buffer solution. As shown in Figure 30.3.1 , the positively charged end of the alkyl ammonium ions bind to the negatively charged silanate ions on the capillary’s walls. The tail of the alkyl ammonium ion is hydrophobic and associates with the tail of another alkyl ammonium ion. The result is a layer of positive charges that attract anions in the buffer. The migration of these solvated anions toward the anode reverses the electroosmotic flow’s direction. The order of elution is exactly opposite that observed under normal conditions.

Figure 30.3.1 . Two modes of capillary zone electrophoresis showing (a) normal migration with electroosmotic flow toward the cathode and (b) reversed migration in which the electroosmotic flow is toward the anode.

Coating the capillary’s walls with a nonionic reagent eliminates the electroosmotic flow. In this form of CZE the cations migrate from the anode to the cathode. Anions elute into the source reservoir and neutral species remain stationary.

Capillary zone electrophoresis provides effective separations of charged species, including inorganic anions and cations, organic acids and amines, and large biomolecules such as proteins. For example, CZE was used to separate a mixture of 36 inorganic and organic ions in less than three minutes [Jones, W. R.; Jandik, P. J. Chromatog. 1992, 608, 385–393]. A mixture of neutral species, of course, can not be resolved.

Micellar Electrokinetic Capillary Chromatography (MEKC)

One limitation to CZE is its inability to separate neutral species. Micellar electrokinetic capillary chromatography overcomes this limitation by adding a surfactant, such as sodium dodecylsulfate (Figure 30.3.2 a) to the buffer solution. Sodium dodecylsulfate, or SDS, consists of a long-chain hydrophobic tail and a negatively charged ionic functional group at its head. When the concentration of SDS is sufficiently large a micelle forms. A micelle consists of a spherical agglomeration of 40–100 surfactant molecules in which the hydrocarbon tails point inward and the negatively charged heads point outward (Figure 30.3.2 b).

Figure 30.3.2 . (a) Structure of sodium dodecylsulfate and (b) cross section through a micelle showing its hydrophobic interior and its hydrophilic exterior.

Because micelles have a negative charge, they migrate toward the cathode with a velocity less than the electroosmotic flow velocity. Neutral species partition themselves between the micelles and the buffer solution in a manner similar to the partitioning of solutes between the two liquid phases in HPLC. Because there is a partitioning between two phases, we include the descriptive term chromatography in the techniques name. Note that in MEKC both phases are mobile.

The elution order for neutral species in MEKC depends on the extent to which each species partitions into the micelles. Hydrophilic neutrals are insoluble in the micelle’s hydrophobic inner environment and elute as a single band, as they would in CZE. Neutral solutes that are extremely hy- drophobic are completely soluble in the micelle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium between the buffer and the micelles elute between the completely hydro- philic and completely hydrophobic neutral species. Those neutral species that favor the buffer elute before those favoring the micelles. Micellar electrokinetic chromatography is used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives.

Capillary Gel Electrophoresis (CGE)

In capillary gel electrophoresis the capillary tubing is filled with a polymeric gel. Because the gel is porous, a solute migrates through the gel with a velocity determined both by its electrophoretic mobility and by its size. The ability to effect a separation using size is helpful when the solutes have similar electrophoretic mobilities. For example, fragments of DNA of varying length have similar charge-to-size ratios, making their separation by CZE difficult. Because the DNA fragments are of different size, a CGE separation is possible.

The capillary used for CGE usually is treated to eliminate electroosmotic flow to prevent the gel from extruding from the capillary tubing. Samples are injected electrokinetically because the gel provides too much resistance for hydrodynamic sampling. The primary application of CGE is the separation of large biomolecules, including DNA fragments, proteins, and oligonucleotides.

Capillary Electrochromatography (CEC)

Another approach to separating neutral species is capillary electrochromatography. In CEC the capillary tubing is packed with 1.5–3 μm particles coated with a bonded stationary phase. Neutral species separate based on their ability to partition between the stationary phase and the buffer, which is moving as a result of the electroosmotic flow; Figure 30.3.3 provides a representative example for the separation of a mixture of hydrocarbons. A CEC separation is similar to the analogous HPLC separation, but without the need for high pressure pumps. Efficiency in CEC is better than in HPLC, and analysis times are shorter.

Figure 30.3.3 . Capillary electrochromatographic separation of a mixture of hydrocarbons in DMSO. The column contains a porous polymer of butyl methacrylate and lauryl acrylate (25%:75% mol:mol) with butane dioldacrylate as a crosslinker. Data provided by Zoe LaPier and Michelle Bushey, Department of Chemistry, Trinity University.