Capillary Electrophoresis Methods

Capillary Electrophoresis Methods

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

Capillary Zone Electrophoresis 

The simplest form of capillary electrophoresis is capillary zone electrophoresis (CZE). In CZE the capillary tube is filled with a buffer solution and, after loading the sample, the ends of the capillary tube are placed in reservoirs containing additional buffer solution. 

Under normal conditions, the end of the capillary containing the sample is the anode, and solutes migrate toward the cathode at a velocity determined by their electrophoretic mobility and the electroosmotic flow. Cations elute first, with smaller, more highly charged cations elut- ing before larger cations with smaller charges. Neutral species elute as a single band. Finally, anions are the last species to elute, with smaller, more negatively charged an- ions being the last to elute.

The direction of electroosmotic flow and, therefore, the order of elution in CZE can be reversed. This is accomplished by adding an alkylammonium salt to the buffer solution. As shown in Figure 12.45, the positively charged end of the alkyl- ammonium ion binds to the negatively charged silanate ions on the capillary’s walls. The alkylammonium ion’s “tail” is hydrophobic and associates with the tail of an- other alkylammonium ion. The result is a layer of positive charges to which anions in the buffer solution are attracted. The migration of these solvated anions toward the anode reverses the electroosmotic flow’s direction. The order of elution in this case is exactly the opposite of that observed under normal conditions.

Capillary zone electrophoresis also can be accomplished without an electroos- motic flow by coating the capillary’s walls with a nonionic reagent. In the absence of electroosmotic flow only cations migrate from the anode to the cathode. Anions elute into the source reservoir while neutral species remain stationary.

Capillary zone electrophoresis provides effective separations of any charged species, including inorganic anions and cations, organic acids and amines, and large biomolecules such as proteins. For example, CZE has been used to separate a mix- ture of 36 inorganic and organic ions in less than 3 minutes.17 Neutral species, of course, cannot be separated.

Micellar Electrokinetic Capillary Chromatography 

One limitation to CZE is its in- ability to separate neutral species. Micellar electrokinetic chromatography (MEKC) overcomes this limitation by adding a surfactant, such as sodium dodecyl- sulfate (Figure 12.46a) to the buffer solution. Sodium dodecylsulfate, (SDS) has a long-chain hydrophobic “tail” and an ionic functional group, providing a negatively charged “head.” When the concentration of SDS is sufficiently large, a micelle forms. A micelle consists of an agglomeration of 40–100 surfactant molecules in which the hydrocarbon tails point inward, and the negatively charged heads point outward (Figure 12.46b).

Because micelles are negatively charged, they migrate toward the cathode with a velocity less than the electroosmotic flow velocity. Neutral species partition them- selves between the micelles and the buffer solution in much the same manner as they do in HPLC. Because there is a partitioning between two phases, the term “chromatography” is used. Note that in MEKC both phases are “mobile.”

The elution order for neutral species in MEKC de- pends on the extent to which they partition 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 ex-tremely hydrophobic are completely soluble in the mi- celle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium be- tween the buffer solution and the micelles elute between the completely hydrophilic and completely hydrophobic neutrals. Those neutral species favoring the buffer solution elute before those favoring the micelles. Micellar electrokinetic chromatography has been used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives.

Capillary Gel Electrophoresis 

In capillary gel electrophoresis (CGE) the capil- lary tubing is filled with a polymeric gel. Because the gel is porous, solutes mi- grate through the gel with a velocity determined both by their electrophoretic mobility and their size. The ability to effect a separation based on size is useful when the solutes have similar electrophoretic mobilities. For example, fragments of DNA of varying length have similar charge-to-size ratios, making their separa- tion by CZE difficult. Since the DNA fragments are of different size, a CGE sepa- ration is possible.

The capillary used for CGE is usually treated to eliminate electroosmotic flow, thus preventing the gel’s extrusion 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 

Another approach to separating neutral species is capillary electrochromatography (CEC). In this technique the capillary tubing is packed with 1.5–3-μm silica particles coated with a bonded, nonpolar stationary phase. Neutral species separate based on their ability to partition between the sta- tionary phase and the buffer solution (which, due to electroosmotic flow, is the mo- bile phase). Separations are similar to the analogous HPLC separation, but without the need for high-pressure pumps. Furthermore, efficiency in CEC is better than in HPLC, with shorter analysis times.