Capillary electrophoresis

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Schematic structure of a capillary electrophoresis apparatus

Capillary electrophoresis (English Capillary Electrophoresis , CE) is an analytical separation method based on electrophoresis .

Electrophoresis is the movement (migration / transport) of charged particles ( ions ) in a (mostly liquid) medium under the influence of an electric field. The migration speeds of different ions depend on their charge, shape and effective size as well as on the solution environment and on the strength of the electric field . Therefore, in the course of an electrophoretic migration, different ions separate. This can be used preparatively and, above all, analytically .

In capillary electrophoresis, this separation takes place in a thin capillary tube in an electrolyte solution . The sample volumes can be in the range of as little as 10 nanoliters (0.01 mm³). Typical analysis times are between 2 and 10 minutes.

Basics

Electrophoretic Mobility

The basics of electrophoresis are described by electrokinetics . The electrophoretic mobility of the mobile charge carriers is decisive for the speed of migration in the electric field .

Electro-osmotic flow

The electroosmotic flow ( EOF ), which usually overlaps the electrophoretic migration, is important for capillary electrophoresis. Its strength depends on the pH of the electrolyte and the charge near the capillary surface. The electroosmotic flow occurs as a result of the interface phenomenon between the inner capillary wall and the electrolyte solution when an electric field is applied.

In contrast to chromatographic processes such as high-performance liquid chromatography , capillary electrophoresis results in a very flat flow profile due to the electroosmotic flow . This results in only a slight broadening of the bands, combined with a higher sharpness of the peaks .

history

The Swedish researcher Arne Tiselius (1902–1971) is regarded as the inventor of electrophoresis . He was the first to use the technology analytically and chemically. In 1948 he was awarded the Nobel Prize in Chemistry for his work . The classical electrophoresis introduced by Arne Tiselius uses gels or paper strips that are soaked with an electrolyte solution. When an electrical field is applied, charged substances are separated by drawing cations to the cathode and anions to the anode , neutral substances do not migrate. Classic gel or paper electrophoresis has two major disadvantages. A quantitative evaluation is only possible with reflectance measurement. B. Proteins can only be examined after staining, and therefore highly error-prone. In order to prevent the gel or the paper strip from drying out, the voltage must not be applied too high, since the Joule heat increases quadratically with the voltage. However, low voltages lead to very long analysis times, so that the analysis time can be several hours for a 10 cm long gel stretch. In order to get the detection and cooling problems under control, attempts have been made to transfer electrophoretic separation to open tubes, as is customary in high-performance liquid chromatography and gas chromatography . However, new problems arose from convection currents in the electrolyte. The first separation in an open glass tube was described by Hjertén in 1967.

Development of capillary electrophoresis

The actual development of capillary electrophoresis began with the pioneering work of Mikkers, Everearts, and Verheggen in the late 1970s. The success was achieved by using thin capillaries made of glass and Teflon with internal diameters between 200 and 500 µm. But today's highly efficient separation performance of capillary electrophoresis could only by used by Jorgenson and Lukacs in 1981, from the gas chromatography known, fused silica capillaries are achieved microns with internal diameters of 50 to 200 micrometers. The more favorable surface / volume ratio of the capillaries greatly reduced the disruptive influence of thermally induced convection , and capillaries made of quartz also enabled the use of detectors such as those used in high-performance liquid chromatography . In recent years, capillary electrophoresis has established itself as a notable alternative to high-performance liquid chromatography, and many of the separation principles of this have been transferred to capillary electrophoresis. Capillary electrophoresis has established itself in analytics primarily due to its high separation efficiency, the ease with which it can be automated and the wide adaptability of the separation conditions. In recent years, capillary electrophoresis has become particularly popular for the analysis of therapeutic proteins . Well-known companies operate the quality control of monoclonal antibodies with it.

Construction of a capillary electrophoresis apparatus

A capillary filled with electrolyte, which is immersed with both ends in vessels (vials) filled with the electrolyte, forms the main component of the capillary electrophoresis apparatus. The high voltage for the separation process is supplied via these electrolyte vessels. For most applications, 20–100 cm long, polyimide- coated fused-silica capillaries with an internal diameter of 50–250 µm are used. The polyimide coating allows for better handling because the capillaries do not break easily. A voltage of up to 30 kV can be applied by a high voltage source. The use of quartz capillaries enables UV detection, but before the capillary is inserted into the strongly colored polyimide film, a detector window through which detection is carried out must be scratched or burned. In addition to the UV detectors, fluorescence detectors , inductive conductivity detectors , electrochemical or radioactive detectors can be used. A combination of capillary electrophoresis with mass spectrometry (MS) in a capillary electrophoresis mass spectrometer is technically far more difficult than with high-performance liquid chromatography, since very small amounts of liquid elute from the capillary . For a coupling with mass spectrometry, the flow usually has to be filled with a "sheath liquid". Despite the technically more demanding coupling, coupling with time-of-flight mass spectrometers results in far higher sensitivities and better possibilities for speciation than with conventional detectors.

Analysis process

In order to introduce the sample into the capillary, the electrolyte container at the capillary inlet is exchanged for a sample container. The sample injection itself can be done in several ways. It can be carried out hydrodynamically by applying pressure to the capillary inlet or by applying a vacuum to the capillary outlet. The injected sample volume is then determined by the size of the pressure or vacuum and by time.

The sample can also be applied hydrostatically (siphon injection). In this case, the sample vessel at the inlet is raised by 5–20 cm or the vessel at the capillary outlet is lowered, creating a hydrostatic flow through the difference in level . In this case, the injected sample volume is determined by the hydrostatic pressure difference and the time. In fact, however, the sample volume, which is in the order of magnitude of only approx. 10 nl, is not determined, but rather the method is calibrated against a standard injected under the same conditions .

Another possibility is electrokinetic injection by applying a voltage . It is mainly used for very dilute samples. The ions are allowed to collect in the electric field at the capillary inlet. Furthermore, in this case the electroosmotic flow (EOF, see below) draws in a certain sample volume.

After the sample has been applied, the sample vessel is replaced by an electrolyte vessel and the electrophoresis voltage is applied. This causes electrophoretic migration and separation of the ionic analytes in the capillary as well as the electroosmotic flow .

While the electro-osmotic flow drives the electrolyte and the sample stored in between through the capillary, the analyte ions migrate ahead of or towards the electro-osmotic flow according to their specific migration speed and thus collect in specific zones. If these zones are driven past the detection window towards the end of the capillary, the detector records them one after the other as ion-specific " peaks ". These detector reactions are recorded by a recorder or a computer and, as in high-performance liquid chromatography, identified according to their relative migration speed and evaluated quantitatively according to their area .

techniques

A wide variety of techniques are used in capillary electrophoresis, one of which is capillary zone electrophoresis (CZE). The sample is applied here as the narrowest possible band. After applying the electric field , each component of the sample moves depending on its mobility, so that in the ideal case, pure zones with only one component are formed. There is a separation based on charge and mobility. Neutral molecules are drawn to the cathode by the electroosmotic flow , but not separated.

With the introduction of micellar electrokinetic chromatography (MEKC), neutral molecules could also be separated. In the case of MEKC, micelles are formed by adding detergents such as sodium dodecyl sulfate (SDS) to the electrolyte. This results in the separation of the neutral molecules according to their distribution between the micelles.

In addition, methods such as capillary gel electrophoresis (CGE) and isoelectric focusing (CIEF) have been developed. In capillary gel electrophoresis, the capillary is coated with a polymeric gel, e.g. B. polyacrylamide filled. This also makes use of molecular sieve effects to improve the separation.

With isoelectric focusing , amphoteric sample components are separated along a pH gradient. A separation occurs because, for example, amino acids as amphoteric compounds only migrate until their isoelectric point is reached, i.e. they are outwardly neutral.

Another possible application is isotachophoresis (ITP). A discontinuous buffer system consisting of lead and end electrolyte is used. Since the mobilities of the supporting electrolyte (highest mobility) and the end electrolyte (lowest mobility) differ, a potential gradient is created when a constant current is applied while observing Ohm's law .

literature

  • Petr Jandik, Günther Bonn : Capillary Electrophoresis of Small Molecules and Ions. VCH, New York NY et al. 1993, ISBN 1-56081-533-7 .
  • Patrick Camilleri (Ed.): Capillary Electrophoresis. Theory and Practice. 2nd edition. CRC Press, Boca Raton FL et al. 1998, ISBN 0-8493-9127-X .

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