Capillary electrophoresis (CE) is a fairly recent separation technique, developed in the 1980s. It offers vast improvements over conventional electrophoresis methods. Indeed, the strong interest of the scientific community in this technique can only be compared to that of liquid chromatography in the 1970s.
Capillary electrophoresis has numerous applications and provides the advantage of high resolution, speed, ease of use, automation and low cost. CE can be applied to a wide range of compounds, ranging from small ions to macromolecules. Numerous CE methods have been developed for pharmaceutical, biological, phytochemical and environmental applications. The quantitative potential of this technique, both in terms of accuracy and robustness, has also been demonstrated.
The instrumentation needed to perform capillary electrophoresis is very simple. Schematically, CE is composed of a silica capillary with both extremities resting in tanks filled with a buffer solution. Capillaries are usually 30-100 cm long and have an internal diameter of 50 or 75 ìm. An electrical field of up to 30 kV may be applied through electrodes immersed in the electrolyte solution in the two tanks.
The electrolyte solution is generally made up of an aqueous buffer with precise pH and ionic strength. It may also contain specific additives (e.g. cyclodextrins, surfactants, organic modifiers) that enhance the separation of molecules. Prior to use, the electrolyte solution is thoroughly filtered and degassed. In order to avoid Joule heating, the current and the voltage are rigorously controlled and electrophoresis takes place in a thermostatic chamber.
The different compounds separated by electrophoresis are generally detected during the run by a UV-visible absorbance detector or a fluorometer positioned at the cathode end of the capillary. A recent, commercially available system makes it possible to couple CE to a mass spectrometer (MS) in real time. Besides adding sensitivity and selectivity, MS also provides structural information about the separated molecules. This coupled technique can thus be used to analyze pharmaceutically active compounds and their metabolites in any biological fluid (e.g. plasma, urine, saliva). The coupling of the two techniques requires the addition of a specific solution to the effluent from the capillary. The mixture is then vaporized, ionized and finally introduced into the mass spectrometer.
There are two main methods to introduce the sample into the capillary: hydrodynamic injection (by pressure, aspiration or siphoning) and electrokinetic injection. The latter method is particularly useful in the detection of pharmaceutical product present in the ppb range. The injection volumes may be as low as a nanoliter and a few microliters of the sample are sufficient to carry out the entire analysis. Because of such small volumes, very expensive and even exotic additives can be used in developing new electrophoresis methods.
In electrophoresis, strong electrical fields allow very rapid separations. Another advantage of capillary electrophoresis is its capacity to generate 400,000 to 1 million theoretical plates. The efficiency increases with higher voltages. As a rule, macromolecules that normally have lower diffusion coefficients than small molecules yield higher efficiencies.
It can be shown that when the CE injection and detection volumes are very small, the widening of the peaks is essentially caused by axial diffusion or Joule heating generated by the electrical field. In other words, a faster migration of the chemical species within the capillary yields finer peaks.
As a diagnostic technique, electrophoresis relies on the difference in the mobilities of two distinct substances subjected to an electrical field. It is thus complementary to liquid chromatography and plays an important role among the different analytical techniques currently available.
Electrophoretic transport phenomena
Capillary electrophoresis is a method for separating the constituents of a liquid mixture based on their different mobilities inside an electrical field. This method applies to molecules that are either positively or negatively charged and which migrate in an electrical field with different velocities. The time-dependent separations of the different constituents in a mixture depend on two principal factors called electrophoretic mobility and electroosmotic flow.
Effective electrophoretic mobility (EP mobility). EP mobility (or migration) refers to the movement of charged molecules in a solution of electrolytes thought to be “immobile”, towards the electrode of the opposite sign. Indeed, a charged molecule in an electrical field is subjected to a force that is proportional to its effective charge (q) and the electrical field which is applied (E). Neutral species are not separated unless there is some association with the ions of the electrolyte itself, leading to different drag forces. Different mobilities are designated as (+) or (-) depending on the charge that is carried. For species that have no net charge, mobility is nil.
Electroosmotic flow (EOF). The second parameter which affects the mobility of the solutes is called electroosmotic flow (or electroosmolarity). It finds its origins in the inner wall of the capillary which is lined with silanol groups. These groups become ionized at pH 2 and above and thus create a negatively charged inner lining. To maintain electrical neutrality the cations from the buffer solution cover this lining, thus creating a second layer. When an electrical field is created, the cations from this double layer start migrating towards the cathode. Their movement drags the buffer solution and creates a flow called the electroosmotic flow. This flow can be controlled by changing the electrical tension, the pH, the type of capillary used and by including specific additives. Because of electroosmotic flow, neutral molecules and anions may move in the direction of the detector which is usually located on the cathode end of the capillary.
Apparent mobility (EM). The apparent mobility corresponds to the effective speed of migration of the molecules inside the capillary. It is the sum of their electrophoretic mobility and electroosmotic flow. Thus, in capillary electrophoresis, the time required for a solute to migrate depends on its electrophoretic mobility and on the mobility of the flow.
In capillary electrophoresis, two principal modalities exist for injecting the sample: hydrodynamic injection and electromigration. Hydrodynamic injection may utilze pressure or vacuum. It cannot be used for capillary gel electrophoresis or in the presence of viscous buffer solutions which require longer injection periods. Pressure injection is the most common method in which the injection volume is limited to 1 to 2% of the total volume of the capillary. Injection may also rely on gravity, a method which consists in simply raising the sample. It allows to adjust the amount to be injected with higher precision than with pressure. The electromigration method is much less common.
Types of electrophoresis
Several types of electrophoresis have been developed, including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), isoelectric focusing, capillary gel electrophoresis and capillary isotachophoresis.