6
December 2010 Figure 5. Megabase multicapillary
surfactant is the anionic detergent is sodium dodecyl sulphate at concentrations above its critical micellar concentration (ca. 8.2mM). Although the efficiency of MEKC is somewhat below that of CZE with typically N<200000 plates/m the separations can be impressive. The presence of micelles in the electrolyte does not compromise any underlying CZE separations and so complex samples containing both charged and un-charged molecules can be analysed with ease. This has been used to good effect in metabolomic screening of human samples 20,21 illustrated in (Figure 4).
and is
By the start of the new millennium only two manufacturers, Agilent and Beckman, were still actively marketing analytical CE systems into small niche markets such as chiral analyses in the pharmaceutical industry and high resolution analyses in the biomedical area e.g. for metabolomic studies. A fresh period of hyperactivity followed from 1995 when exceptional separations of pharmaceuticals using capillary electrochromatography (CEC) were published. In this technique the capillary was packed with an appropriate HPLC packing material and the eluent was driven down the capillary using the generated EOF. All this could be achieved using only slightly modified versions of the available CE systems. However CEC proved even less reliable in practice then CE itself and few workers have continued to use the technique.
Around 2000 this author would often ask analytical colleagues to name the biggest seller of CE systems. Invariably one of the two names given above would be quoted and I’d delight in telling them they were very wrong.
The biggest companies were those manufacturing DNA sequencers. Whereas proteins had proved a considerable challenge to separate in a capillary format DNA had not. The initial concept had been to use the slab gel approach and form acrylamide type gels within the capillary but this proved both difficult and unreliable. The much simpler method of using a viscous polymer in the electrolyte was remarkably effective in resolving DNA fragments. This approach in combination with sequencing chemistries using fluorescent dye terminators, laser induced fluorescent detection and multiple capillaries running in parallel CE was to be found in the majority of high throughput DNA sequencers such as the ABI 3700. These systems require speed and all that is needed
qualitative results. It could be argued that without CE we would still be sequencing the human genome rather than having read the first version 10 years ago. The role of CE in the human genome project has been covered by Zhang and Dovichi . Today the need is for even faster sequencing in areas such as human medical research and forensic analysis. The original 16 capillary systems have been replaced with 96 capillary systems holding stacks of 96 well plates and 384 capillary systems are becoming available from a number of companies (Figure 5). The recently introduced megaBACE 4000 from GE Healthcare uses 384 parallel capillaries and can generates over 2.8 million bases per day. The latest development focus on removing the fluorescence detection and methods based on pyrosequencing using enzymes with chemiluminescence detection are being developed. Such CE based systems are currently being incorporated into small and inexpensive sequencers
Protein separations by CE have proved difficult due to the binding of many proteins to the silica wall of the capillary. The possible to exception to this broad statement is the ready separation of human serum/plasma proteins. The ten or so common proteins in plasma are readily separated by CE using simple borate buffers with detection at ca. 210 nm (Figure 6). The separation is rapid and gives direct quantitation unlike the tradition agarose gel methods, which are slow and require staining and densitometric measurements. Even standard analytical systems are capable of running 10 plasmas per hour. A number of years ago Beckman developed at 8 capillary clinical protein
Figure 6. CZE separation of the proteins in normal human serum.
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