Optics: measuring and testing – By electrophoresis
Patent
1994-10-07
1997-05-06
Pham, Hoa Q.
Optics: measuring and testing
By electrophoresis
204452, 204603, 73 6158, 210656, 210 85, 2101982, G01N 2726
Patent
active
056276435
DESCRIPTION:
BRIEF SUMMARY
In the bioscientific field there is a great need of being capable of analysing complex mixtures of biomolecules. Examples are the analysis of metabolites, proteins, antibodies and drugs in clinical analysis. Another example is the analysis of protein composition in the fermentation of proteins specified by genetic engineering.
A usual way of performing bioanalyses is to separate a sample into its constituents and then quantify the constituents. Conventional separation methods are chromatography and electrophoresis. Electrophoresis has a strong position just for analysis. The reason is that the method is high-resolving, which among other things means that many components can be analysed in one and the same run. In combination with suitable detection methods for the separated substances, the method may also be made very sensitive. On the other hand, the method is generally slow, typical separation times being in the range of 1-10 hours.
For the last couple of years a special form of electrophoresis has attracted much attention, viz. capillary electrophoresis. The electrophoretic process takes place in thin capillaries of quartz (inside diameter about 0.005-0.1 mm). The thin capillaries make it easy to cool off the Joule heat that is generated during the process of the electrophoresis. It is therefore possible to use very high field intensities in the electrophoretic separation, e.g. 300 V/cm. Since the high field strength gives a very rapid migration rate, the separation times become favourably short with capillary electrophoresis. In normal electrophoresis, temperature gradients easily arise in the separation bed. Temperature gradients lead to a heavily impaired resolution of the sample components. This problem is eliminated by the capillary electrophoresis technique, where the thin capillaries are easy to cool and therefore do not lead to problematic temperature gradients. Consequently, also the resolving power is very good with capillary electrophoresis. An illustrative review article has been written by H. Carchon and E. Eggermont (International Chromatography Laboratory, Vol. 6, pages 17-22, 1991).
The detection and quantification of mixtures separated by capillary electrophoresis are usually performed with a UV/Vis detector or fluorescence detector (a review of different detection principles for capillary electrophoresis has been written by D. M. Goodall, D. K. Lloyd and S. J. Williams in LC-GC, Vol. 8, No. 10, 1990, pages 788-799). These detectors are always placed at one end of the capillary. Usually a light beam is made to pass perpendicularly through the capillary, and a detector measures the amount of light that has passed or the amount of fluorescence that has been induced. The separated substances are thereby detected one after the other as they pass the detector. This means that the electrophoresis must continue until all the substances have passed the detector. Often the number of substances in the sample is not known, and the electrophoretic run must therefore proceed considerably longer than actually necessary in order to ensure that all the sample components have really passed the detector. A limiting factor of capillary electrophoresis is thus seen here, viz. the serial detection principle, i.e. the sample components being detected one after the other. If instead a detection principle is provided where the whole capillary is examined momentarily by a special detector, a parallel detention principle is obtained where all the components are detected simultaneously. The present invention discloses just such a principle and the construction of suitable apparatus.
With the present invention, a sample may be separated into components in a thin capillary. At short intervals during the separation, the whole, or at least a large part, of the capillary may be simultaneously irradiated and either the emitted or absorbed light may be detected, thus creating successive momentary images of the separation pattern. The detection of the light may be based on, but is not limited to, fluorescence and light s
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Birnbaum Staffan
Johansson Jonas
Larsson Per-Olof
Miyabayashi Akiyoshi
Mosbach Klaus
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