Electrophoretic separation methods are based on the different rates of migration of the individual components of a test sample in a carrier medium when an electric field is applied. A very widely used method is capillary electrophoresis in which a carrier medium and a sample to be tested are transported in a capillary system which comprises a capillary separating path between the ends of which the electric field is applied. The transport of the carrier medium in the capillary system and the injection of the sample to be tested into the carrier medium can be carried out with the aid of pumps and valves or using electric fields which are suitably applied in various portions of the capillary system. The individual components of the sample injected into the carrier medium migrate at different rates in the electric field of the separating path, with the result that the sample is separated. The individual components can be determined with the aid of a detector connected to the capillary separating path. For the simultaneous analysis of different samples there have also been proposed separating arrangements having several parallel capillaries (Anal. Chem. 1992, 64, 967-972).
In DNA sequencing, for example, gel-filled capillaries are used as the separating path. In that separating method the carrier medium, i.e. the gel, is not transported; instead only the sample injected into the gel migrates in the applied electric field. A typical separating performance (which is also referred to as the theoretical separating step number) of an electrophoretic separating system using such gel-filled capillaries is, for example, about 250 peaks in a period of 30 minutes.
U.S. Pat. No. 4,908,112 proposes the miniaturisation of branched capillary systems including the separating path. The capillary system is arranged on a semi-conductor chip. The transport of the carrier medium and the injection of the sample to be separated are effected with the aid of electric fields that can be switched between individual path portions of the capillary system. The dimensions of the channel system are very small but the field strengths that can be achieved are very high. Consequently only very small amounts of carrier medium and very small sample volumes are required. In addition, the separating method can be carried out very quickly at the high voltages applied, which are typically about 30 kV.
Another very widely used electrophoretic separating method is gel electrophoresis. That separating method, in which the separation of the sample into its constituents is effected not in solution but in a stationary carrier material, a gel, is also known as electropherography. In the electropherographic method the sample to be separated is applied as a strip preferably in the centre of a carrier material steeped in buffer (the pherogram) and an electrical voltage is applied to the ends of the carrier material. The sample is separated in accordance with the direction of migration and the rate of migration of the individual components. The differently charged components migrate to the respective oppositely charged poles, while the neutral components remain at the point of application. In a continuous separating method a buffer solution flows through a vertical plate of carrier material. The sample is added as near as possible to the upper end of the plate. The electrophoretic separation is brought about by an electric field applied perpendicularly to the flow of buffer.
Gel electrophoresis is an established separating method for charged biopolymers. Polyacrylamide gels (PAGE) are frequently used for the separation. The pore size of the polyacrylamide gels allows separation in accordance with the charge and the steric hindrance of the sample molecules in the gel. If sodium dodecyl sulfate (SDS) is added, good correlation is obtained between the migration distance of the separated sample molecules and the corresponding molar mass, which is, however, independent of the charge of the molecules. Isoelectric focussing (IEF or IF) as a preliminary stage before SDS-PAGE gel electrophoresis makes it possible also to separate many extremely complex substance mixtures.
A moderately well established further development of gel electrophoresis is so-called 2D gel electrophoresis in which a sample is separated in two dimensions (2D) in accordance with different criteria. Such a 2D gel electrophoresis separating arrangement is described, for example, in A. T. Andrews, "Electrophoresis, Theory, Techniques and Biochemical and Clinical Applications", Clarendon Press, Oxford 1986, pages 223-230. That two dimensional separating method is used especially as a combination of isoelectric focussing in the first dimension and gel electrophoresis, for example SDS-PAGE gel electrophoresis, in the second dimension. The resulting gel pattern provides in the first dimension information relating to the isoelectric point of the component in question and in the second dimension information relating to the molar mass of that component. A typical separating performance in 2D gel electrophoresis is a peak capacity of about 10 000 in a time period of more than 2 hours.
Although it is possible to obtain very high separating performances with 2D gel electrophoresis, a disadvantage of that method is that it is very slow. First of all the sample must be separated in the first dimension on a first gel. The first gel then has to be brought together with a second gel in which the separation in the second dimension is to take place, which is usually a laborious operation. The long analysis time results in the diffusion of the separated components in the free gel, which can lead to an undesirable broadening of the bands. The electrical voltage necessary for separation in the gel can be increased only to a limited extent and is typically about 2 kV. At higher voltages Joule effect heating occurs, which can result in the decomposition of the gel and the sample.
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