Capillary electrophoresis

Capillary electrophoresis (English Capillary Electrophoresis, CE) is a community based on the electrophoresis analytical separation method.

Electrophoresis refers to the movement ( migration / transport) of charged particles (ions ) in a ( usually liquid ) medium under the influence of an electric field. The migration rates of various ions depend on their charge, shape and effective size as well as the solution environment and the strength of the electric field. Therefore, it comes in the wake of an electrophoretic migration for separation of different ions. This can be used analytically synthetically and especially.

In capillary electrophoresis, this separation takes place in an electrolyte solution in a thin capillary tube. The sample volumes may be in the range of 10 nano liters ( 0.01 mm ³). Typical analysis times are between 2 and 10 minutes.

Basics

Electrophoretic mobility

The basics of electrophoresis are described by electrokinetics. Critical to the migration rate in an electric field, in particular, the electrophoretic mobility of the mobile charge carrier.

Electroosmotic flow

Important for capillary electrophoresis is the electroosmotic flow (EOF ), which is usually superimposed on the electrophoretic migration. Its thickness is dependent on the pH of the electrolyte and the charge in the vicinity of the capillary. EOF occurs as a result of the interfacial phenomenon between the inner capillary and the electrolyte solution when an electric field.

Unlike chromatographic methods such as HPLC resulting in capillary, due to the electroosmotic flow of a very flat flow profile. This leads to only a slight broadening of the bands, coupled with a higher sharpness of the peaks.

History

As the inventor of the electrophoresis, the Swedish researcher Arne Tiselius (1902-1971) is considered. He has used the technique first on analytical chemistry, he was awarded the 1948 Nobel Prize in Chemistry for his work. In classical electrophoresis introduced by Arne Tiselius using gels or strips of paper impregnated with an electrolyte solution. By applying an electric field it came to the separation of charged substances by cations were attracted to the cathode and anions to the anode, neutral substances are not migrated. The classic gel or paper electrophoresis has two major drawbacks. A quantitative evaluation is only possible with remission measurement, eg Proteins after coloration, and therefore, significant errors can be studied. To prevent drying of the gel and the paper strip, not too much voltage may be applied, because the Joule heat increases with the square of voltage. However, low voltages result in very long analysis times, so that for a 10 cm long controlled system, the analysis time can often be several hours. To obtain the detection and cooling problems in the handle, an attempt was made to transfer the electrophoretic separation of open pipes, as in the usual HPLC and GC. However, new problems arose by convection currents in the electrolyte. The first separation in an open glass tube was described in 1967 by Hjertén.

Development of capillary electrophoresis

The actual development of capillary electrophoresis started with the pioneering work of Mikkers, Everearts and Verheggen the late 1970s. This success was achieved by the use of thin capillaries made ​​of glass and Teflon with inner diameters between 200 and 500 microns. The well-known highly efficient separation performance of capillary electrophoresis (CE) were known by 1981 used by Jorgenson and Lukacs, from the GC but only fused silica capillaries can be achieved with internal diameters of 50 to 200 microns. The more favorable surface / volume ratio in the capillaries reduced the disturbing influence of convection thermally induced strong and capillaries made ​​of quartz also enabled the use of detectors, such as those used in HPLC. In recent years, CE has established itself as a significant alternative to HPLC, also were many of the HPLC separation principles applied to the CE. Established has the CE in the analysis mainly due to the high separation efficiency, good automation and the wide adaptability of the separation conditions. In recent years, capillary electrophoresis has particularly enforced for the analysis of therapeutic proteins. Well-known companies operate the quality control of monoclonal antibodies that.

Building a CE apparatus

A capillary filled with electrolyte, which with both its ends filled with the electrolyte vessels ( vials ) emerges, constituting the main component of the CE apparatus. This electrolyte vessels, the high voltage for the separation process is fed. In most applications using 20-100 cm long, polyimide-coated fused silica capillary with an inner diameter of 50-250 microns. The polyimide coating allows for better handling because the capillaries do not break so easily. By a high voltage source, a voltage of up to 30 kV to the cathode or anode can be applied. Through the use of quartz capillary UV detection is possible, but needs to be scratched or burned a detector window in the highly colored polyimide film is detected through which prior to the application of the capillary. In addition to the UV detectors fluorescence detectors, inductive conductivity detectors, electrochemical detectors can be used, or radioactive. A combination of CE with mass spectrometry ( MS) designed technically far more difficult than in HPLC because elute from the capillary very small amounts of liquid. For a connection to the mass of the flow must usually be filled by a "sheath -liquids ". Despite the more sophisticated coupling arising from coupling with TOF mass spectrometers much higher sensitivities and better ways to speciation than with the conventional detectors.

Analysis cycle

To introduce the sample into the capillary, the electrolyte vessel is exchanged for a sample vessel to the capillary inlet. The sample injection itself may be made in several ways. It can be hydrodynamically performed by applying pressure at the capillary inlet or by applying a vacuum at the capillary outlet. The injected sample volume is then determined by the amount of pressure or vacuum and through time.

The sample can be applied hydrostatic ( Siphoninjektion ). In this case, the sample container at the inlet is increased by 5 to 20 cm and the container is lowered at the capillary outlet and as generated by the difference in level, a hydrostatic flow. In this case, the injected sample volume is determined by the hydrostatic pressure difference, and time. But in fact is not the sample volume of the order of only about 10 L, is determined. Rather, the method against a standard injected under the same conditions is calibrated.

A further possibility is the electrokinetic injection by applying a voltage. It is used especially for very dilute samples. Involves leaving the ions accumulate on Kapillareneinlass in the electric field. Also in this case draws the electroosmotic flow ( EOF, see below) a certain sample volume.

After application of the sample, the sample container is again replaced by an electrolyte vessel and the applied electrophoretic voltage. This causes an electrophoretic migration and separation of ionic analytes in the capillary and the EOF.

During the EOF drives the electrolytes and the intervening stored sample through the capillary, the analyte ions migrate according to their specific migration rate of the EOF advance or counter and collect as in specific zones. If these zones driven past the end of the capillary at the detection window, the detector takes them up one after another as an ion- specific " peaks ". This detector responses are recorded by a chart recorder or a computer and identified by HPLC analogously as according to their relative migration velocity and quantitated according to their surface area.

Techniques

In the CE various techniques are used, one of which is the Capillary zone electrophoresis (CZE ). The sample application takes place here as narrow as possible. After applying the electric field, each component of the sample moved because of their mobility, so that trained ideally pure zones with only one component. There is a separation due to the charge and mobility. Neutral molecules are indeed drawn by the electroosmotic flow towards the cathode, but not separated.

By introducing the mizellarelektrokinetischen chromatography ( MEKC ) and neutral molecules could be separated. In the case of the MEKC micelles by the addition of detergents such as sodium dodecyl sulfate (SDS ) are formed for the electrolyte. This results in the separation of neutral molecules according to their distribution among the micelles.

Furthermore, methods were developed such as the capillary gel electrophoresis (CGE ) and isoelectric focusing ( CIEF ). In the capillary, the capillary with a polymer gel, for example, Polyacrylamide filled. Thus, in addition to uses Molekularsiebeffekte to improve the separation.

In isoelectric focusing amphoteric sample components along a pH gradient to be separated. There will be a separation, as for example amino acids as amphoteric compounds only while walking, to its isoelectric point is reached, ie they are neutral to the outside.

Another possible application is the isotachophoresis (ITP ). Here, a discontinuous buffer system of control and end electrolyte used. Since the mobilities of supporting electrolyte (highest mobility) and end electrolyte differ (lowest mobility) forms when applying a constant current in accordance with Ohm's law to a potential gradient from.