5


electrophoresis but its use for the separation of nucleic acids was very limited since they have relatively constant charge to size ratio. RNA had first been separated on agar gels in 196412


and the still widely used background


electrolyte of TRIS-borate-EDTA (TBE) was introduced in 196813


. The separation of DNA


by gel electrophoresis had to wait a few more years and was probably first reported in 1971 by Danna and Nathans14


. A classic paper by


Gilbert and colleagues in 1974 reported the use of TBE buffers for the base specific sequencing of DNA fragments following chemical cleavage15


Figure 3. 2-D (IEF-SDS-PAGE) separation of serum proteins from a patient with osteoarthritis


improvement in the electrophoresis of proteins was the discovery of resolving power of gels made of starch in 19556


. Oliver


Smithies (Nobel Laureate 2008), a native of Halifax, Yorkshire, remembered that his mother used starch on wash days and it set to a gel. He showed that proteins could be readily separated on slabs of starch gels and there was evidence that the separation was molecular size dependent. In 1956 Smithies7 introduced the two dimensional separation of serum proteins: first separating them on paper followed by a starch gel. Many, still important, variants on gel electrophoresis such as immuno-electrophoresis were developed around this time. A major breakthrough occurred with the introduction of synthetic gels synthesised from polyacrylamide in 19598 and their use to “size” proteins in SDS-PAGE electrophoresis in 19679


. The use of SDS-


PAGE gels in a 2-D combination with isoelectric focussing of proteins was developed by a number of groups simultaneously3


given to O’Farrell10


to volume ratio i.e. by using a capillary, the heat generated by electrophoresis was readily lost. Very high voltages could then be employed to enable high speed analyses with on-line detection and both exceptional resolution, and enhanced sample sensitivity.


. This became a standard


technique in DNA sequencing until the arrival of modern sequencers in the late 1990s.


Planar separations of serum proteins on agarose gels continues to be a standard technique in many clinical laboratories. It is a skilled task and although throughput is important it is not a method needing to generate instant results nor is absolute quantitation important. Similar considerations apply to the separation of haemoglobins in the characterisation of haemoglobinopathies and thalassemias, which is usually done on cellulose acetate strips. Clinical laboratories have used semiautomatic planar electrophoresis system for some years. Such equipment has been supplied by companies such as Bio-Rad, Hoeffer etc. and high throughput fully automated clinical electrophoresis equipment is available from Sebia (France).


but the credit is most often . The isoelectric-focussing


step was much improved with the introduction of immobilised pH gradients (IPG strips) in 198211


and their commercialisation in the


1990s. Although prone to many difficulties it is this 2-D approach that still forms the basis of Proteomics in many laboratories.


However electrophoresis on slab gels and similar planar formats was a qualitative technique and even the best computerised digitisation methods can only make it a poor semi quantitative methodology. So with the commercial development of first gas chromatography and then HPLC from about 1965 electrophoresis faded as an analytical method for small molecules. Gel electrophoresis continues to be used for protein analyses especially clinical diagnostic laboratories. Nucleotides and nucleosides were readily separated by paper


Capillary Electrophoresis The major bugbear with all electrophoresis is the generation of Joule heat with its many detrimental effects on electrophoretic separations and resolution. This was effectively overcome with the introduction of capillary electrophoresis by Jorgenson and colleagues. In three classic papers 16,17,18 published in 1981 Jim Jorgenson demonstrated that by increasing the surface


The electroosmotic flow generated by the silanol groups of the polyimide coated silica capillary’s inner surface gives an efficient and fast plug flow to the electrolyte that is in contrast to the parabolic flow found in pumped LC columns and so helps generate very efficient peak shapes . CE is therefore characterised by its ability to resolve using a high applied d.c. voltage (with field strengths up to 500 V/cm), the charged components of complex aqueous samples with very high resolution (N>1 million plates/m). Unlike traditional methods CE performs with levels of analytical precision similar to HPLC but typically uses less than 10 nl of sample. By the early 1990s nearly every manufacturer of HPLC equipment had a CE system in their catalogue. Thousands of papers using CE were being published annually but the original hype from the companies and many academics was beginning to fade. Problems with migration time irreproducibility leading to poor quantitation were commonplace, sensitivities did not match even those of standard HPLC systems and surprisingly the separation of proteins was proving very difficult.


Very shortly after Jorgenson’s first publications Terabe19


introduced a novel variant on CE which enabled the separation of hydrophobic compounds by a pseudoelectrophoretic mechanism. In this variant called micellar electrokinetic chromatography (MEKC) non- polar compounds distribute themselves between an electrosmotically driven mobile phase and the charged surfactant micelles electrophoretically migrating in the opposite direction. In the commonest mode the


Figure 4. U.V. absorbing compounds in human urine separated by sulphated-β-cyclodextrin modified MEKC.


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