Capillary electrophoresis

Capillary electrophoresis (CE) is a family of related techniques that began to gain popularity in the late 1980s, when commercial instrumentation became available. It employs narrow bore (20-100 /¿m) fused silica capillaries, externally coated with plastic (polyimide) for increased durability in lengths of 10-100 cm. (Figure 3.33). Although high voltages (up to 30 000 V) are used, the heat is dissipated quickly because of the relatively large surface area and cooling is not normally required.

High voltage

Figure 3.33 Capillary electrophoresis. The sample is introduced through the inlet, washed in with a wash buffer and then separated under the influence of a potential difference between the two electrodes. The zones are monitored as they pass through the detector and the data captured and computed.

High voltage

Figure 3.33 Capillary electrophoresis. The sample is introduced through the inlet, washed in with a wash buffer and then separated under the influence of a potential difference between the two electrodes. The zones are monitored as they pass through the detector and the data captured and computed.

The sample is usually introduced into the capillary by dipping it into the sample vial and applying a slight positive pressure. This results in a small volume of the sample entering the capillary. Depending upon the pressure and the time, the amount can be carefully controlled. The sample is then moved into the capillary as a zone and the entry to the capillary cleaned at the same time by drawing a set volume of a wash buffer. Alternative techniques include suction, siphoning and an electrokinetic technique, but all achieve the same result. The bands that separate are detected while they are still in the capillary, usually by absorbance, either in the ultraviolet or visible region of the spectrum, sometimes involving a diode array. Alternatively fluorescence or refractive index detectors may be employed and some instruments link the capillary to a mass spectrometer.

Capillary electrophoresis has been shown to have several advantages over other separation techniques and since its introduction the number of applications described for it has risen rapidly. It lends itself to the analysis of a wide range of ionic and neutral molecules of varying size and shape. Analysis times are similar to those for HPLC, typically being less than 30 minutes, but separation efficiency is often superior and theoretical plate numbers of greater than 1 X 106 are typical. Nanolitre sample volumes are used, a factor of great importance when limited amounts are available. Reagent costs are low because only microlitre volumes are required for each analysis. The capillaries must be appropriately conditioned or regenerated before use and should be flushed with water and air for storage. Modern instruments can be programmed to perform all the steps in the analysis from the introduction of the sample through to the interpretation of the results, which are presented as a series of peaks on a chart recorder, and preparation of the capillary for the next sample.

There are several modes of operation of CE which rely on variations of the principles of electrophoretic separation.

> Capillary /on* eec trophoresis is based on conventional electrophoresis of the analyte.

Free solution electrophoresis (capillary zone electrophoresis). This was the first type of capillary electrophoresis to be developed. Migration of the analytes through a buffer solution, which sometimes contains chemical additives to enhance the separation, is affected by their net charge and the elec-troendosmotic flow. The magnitude of the electroendosmotic effect is due to negatively charged silanol groups on the capillary wall, which attract positively charged ions from the buffer, and the overall effect is the movement of the buffered solution towards the cathode. Endosmosis is particularly evident with buffers of high pH and low concentration. A variety of internal polymeric coatings can be applied to the capillaries to modify the electroendosmotic effect and thus extend the applications of the method.

► Micellar electrokinetic chromatography is based on the effects of the interaction of the analyte with surfactant micelles on the electrophoretic process.

Micellar electrokinetic capillary chromatography (MEKC or MECC) This is used to measure small neutral or charged molecules and is applicable to a wide range of analytes including drugs, nucleosides, peptides and vitamins. Surfactants such as sodium dodecyl sulphate (SDS) are added to the buffer solution to produce anionic aggregates called micelles. The surfactants have long-chain hydrophobic tails (between 10 and 50 carbon units) and hydrophilic heads which, when bound in the micelles, point outwards into the solution. Separation depends on the interaction of the analytes with these charged micelles, which modifies their electrophoretic mobility. When negatively charged surfactants such as SDS are used, cationic analytes are strongly attracted to the micelles in a manner that can be likened to the situation in ion-pair chromatography, and the overall negative charge of the micelle is reduced.

Anions and uncharged analytes tend to spend more time in the buffered solution and as a result their movement relates to this. While these are useful generalizations, various factors contribute to the migration order of the analytes. These include the anionic or cationic nature of the surfactant, the influence of electroendosmosis, the properties of the buffer, the contributions of electrostatic versus hydrophobic interactions and the elec-trophoretic mobility of the native analyte. In addition, organic modifiers, e.g. methanol, acetonitrile and tetrahydrofuran are used to enhance separations and these increase the affinity of the more hydrophobic analytes for the liquid rather than the micellar phase. The effect of chirality of the analyte on its interaction with the micelles is utilized to separate enantiomers that either are already present in a sample or have been chemically produced. Such pre-capillary derivatization has been used to produce chiral amino acids for capillary electrophoresis. An alternative approach to chiral separations is the incorporation of additiveis such as cyclodextrins in the buffer solution.

> Capillary gel electrophoresis is based on differences in analyte size.

Capillary gel electrophoresis

This technique is used for the analysis of large molecules such as proteins and nucleic acids. The molecular sieving property of the gels is important in the separation process. The terms 'chemical gel' and 'physical gel' are often used to describe the gel matrices. Chemical gels are cross-linked with relatively high viscosity and have a defined pore structure. Polyacrylamide comes into this category. Physical gels are solutions of entangled polymers, e.g. alkylcelluloses. They have relatively low viscosity and the contents of the capillary can be easily flushed out after use. The concentration and chain length of the linear polymers determines the separation characteristics of the gel solution.

Capillary procedures offer several advantages, including speed, resolution, sensitivity and technical simplicity, compared with the traditional methods on which they were based. An added advantage for iso-electric focusing in capillaries is the fact that it can be performed without a gel, but a coating on the internal surface of the capillary is usually required to reduce electroendosmosis. Similarly, isotachophoresis can be conveniently performed in capillary electrophoresis apparatus.

> Capillary electrochro-matography is based on the effect of interaction of the analyte with the stationary phase on its electrophoretic mobility.

Capillary electrochromatography (CEC)

This is a more recently developed technique which is a hybrid between HPLC and capillary electrophoresis. The capillary is packed with HPLC media and the mobile phases are aqueous buffers. A voltage is applied to generate an electroendosmotic flow and the analytes separate by interaction with the stationary phase and electrophoretic forces; no pump being required as for HPLC. Improved separation efficiencies have been reported.

Self test questions

Section 3.3

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