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December 2010
200 years of Electrophoresis By David Perrett
William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 7BQ U.K.
This brief overview outlines the development of electrophoresis from its first observation some 200 years ago via conventional gels for macromolecule separation and capillary electrophoresis (CE) to current developments centred around lab-on-chip. By definition electrophoresis separates ionic molecules so it is ideal for the separation of simple ions to macromolecules, which are mostly ionic in nature. Most important classes of small biomolecules e.g. amino acids, nucleotides and sugars are highly charged and are easy to separate by electrophoresis. However prior to the development of CE, the application of electrophoresis was limited since it required indirect detection which was at best only semi quantitative and HPLC came to dominate their measurement. Macromolecules, such as RNA, DNA and protein, are readily separated by electrophoresis and conventional electrophoresis still dominates their separation.
Historical background Today electrophoresis remains a very important, if somewhat neglected, analytical technique and is now seen to have three dominant modes i.e. planar, capillary and nano separation formats. However it is just over 200 years since Ferdinand Frederic Reuss published his observations of the migration of colloidal clay particles when an electric field was applied to the solution in which they were suspended1
. In the same experiment he also
found that there was an opposite flow of water (electroosmosis) associated with the movement of the clay particles. These observations are considered to be the origins of what we now call electrophoresis. In 1816 Porret quantified the flow of water (electroosmosis) through filter paper impregnated with egg albumin. Within a few years of Reuss’s observation the movement of coloured proteins, such as haemoglobin, had been observed. The early history of electrophoresis has been told by Righetti2
its relevance to the discovery and analysis of proteins has been reviewed by Perrett3
.
However these early findings were little more than scientific curiosities requiring physico- chemical investigation and went under the name cataphoresis. Many laboratory suppliers sold apparatus to demonstrate these phenomena (Figure 1). The change of name came in 1909 when Michaelis suggested that the name of the technique should be changed to electrophoresis which is derived from the Greek elektron meaning amber (i.e. electric) and phore meaning bearer4
.
The use of electrophoresis for the analysis of complex protein mixtures had to await the work of Arne Tiselius from the Karolinska in Sweden during the 1930s. Although his
by Joule heating and so only low voltages can be used. Elaborate high voltage systems with forced cooling were manufactured by companies such as Miles HiVolt, Shandon and Locarte in the UK (Figure 2) and Savant and Gilson in the USA. They were very large instruments that typically used 3 feet square Whatman filter paper sheets and employed 10 kV at 100 mA, a potentially lethal combination: in 1965 a graduate student at Brown University USA died from electrocution when a short circuit occurred in the high voltage electrophoresis electrical system being used.
Figure 1. Cataphoresis equipment in the Griffin Chemical Supplies Catalogue 1908
and
apparatus was complex and the detection principle rather obscure, Tiselius was able to separate the five most abundant proteins that occur in human serum with relative ease and quantify their levels in both normal and some disease states. Tiselius was awarded a Nobel Prize in 1948 for his work on protein separation by electrophoresis.
From World War Two until the late 1960s the dominant separation techniques were paper chromatography for most uncharged analytes and paper electrophoresis for charged analytes such as amino acids. For both techniques indirect detection methods dominated with colour reactions being essential, e.g. ninhydrin to reveal amino acids and peptides. Electrophoresis on paper sheets followed by staining with dyes such as bromophenol blue became established for the clinical analysis of serum proteins. Paper was later replaced by more robust sheets of cellulose acetate and later nitrocellulose and Ponceau S dye became the most popular stain. Speed of separation of small ionic molecules by planar electrophoresis is limited
Archer Martin and co-workers, although much better known for the development of chromatography, did much novel work on electrophoresis including using agar gels as an electrophoretic medium5
. The use of
agarose, a chemically defined variety of agar that dissolves readily in hot buffer solutions and then sets on cooling, is still commonly used for DNA separations. A major
Figure 2. Cover of a Locarte of London High voltage electrophoresis catalogue of ca.1968. The whole system cost £898.
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