Capillary electrophoresis separation process

Electrophoretic methods are widely used alternatives for the analytical determination of the enantiomeric purity of chiral compounds [194]. Due to the high elTi-ciency of capillary electrophoresis, separations can be achieved even when very low selectivities are observed. At a preparative scale, these methods are well established for the purification of proteins and cells [195] but there is very little published on enantioselective separations. Only recently, some interest in chiral preparative applications has been manifested. Separation of the enantiomers ofterbu-taline [196] and piperoxan [197] have been reported by classical gel electrophoresis using sulfated cyclodextrin as a chiral additive, while the separation of the enantiomers of methadone could be successfully achieved by using free-fluid isotachophoresis [198] and by applying a process called interval-flow electrophoresis [199]. 

Enantioresolution in capillary electrophoresis (CE) is typically achieved with the help of chiral additives dissolved in the background electrolyte. A number of low as well as high molecular weight compounds such as proteins, antibiotics, crown ethers, and cyclodextrins have already been tested and optimized. Since the mechanism of retention and resolution remains ambiguous, the selection of an additive best suited for the specific separation relies on the one-at-a-time testing of each individual compound, a tedious process at best. Obviously, the use of a mixed library of chiral additives combined with an efficient deconvolution strategy has the potential to accelerate this selection. 

At present moment, no generally feasible method exists for the large-scale production of optically pure products. Although for the separation of virtually every racemic mixture an analytical method is available (gas chromatography, liquid chromatography or capillary electrophoresis), this is not the case for the separation of racemic mixtures on an industrial scale. The most widely applied method for the separation of racemic mixtures is diastereomeric salt crystallization [1]. However, this usually requires many steps, making the process complicated and inducing considerable losses of valuable product. In order to avoid the problems associated with diastereomeric salt crystallization, membrane-based processes may be considered as a viable alternative. 

Capillary electrophoresis offers several useful methods for (i) fast, highly efficient separations of ionic species (ii) fast separations of macromolecules (biopolymers) and (iii) development of small volume separations-based sensors. The very low-solvent flow (l-10nL min-1) CE technique, which is capable of providing exceptional separation efficiencies, places great demands on injection, detection and the other processes involved. The total volume of the capillaries typically used in CE is a few microlitres. CE instrumentation must deliver nL volumes reproducibly every time. The peak width of an analyte obtained from an electropherogram depends not only on the bandwidth of the analyte in the capillary but also on the migration rate of the analyte. 

On the other hand, the most severe constraint of CL analyses is their relatively low selectivity. One major goal of CL methodologies is thus to improve selectivity, which can be accomplished in three main ways (1) by coupling the CL reaction to a previous, highly selective biochemical process such as an immunochemical and/or enzymatic reaction (2) by using a prior continuous separation technique such as liquid chromatography or capillary electrophoresis or (3) by mathematical discrimination of the combined CL signals. This last approach is discussed in Sec. 4. 

Some coupled systems allow measurement of the main N and P forms (nitrate, ammonia and orthophosphates) [22,27,29], among which is a system based on membrane technology in combination with semi-micro continuous-flow analysis (pCFA) with classical colorimetry. With the same principle (classical colorimetry), another system [30] proposes the measurement of phosphate, iron and sulphate by flow-injection analysis (FIA). These systems are derived from laboratory procedures, as in a recent work [31] where capillary electrophoresis (CE) was used for the separation of inorganic and organic ions from waters in a pulp and paper process. Chloride, thiosulphate, sulphate, oxalate,... 

Development of batch process in 1987, coupled with fluorescent dideoxy-terminator labeling on target DNA, has allowed determination of fluorescence-tagged DNA sequences, separated on high-resolution slab-gels and more recently separated by capillary electrophoresis. Both separation methods are capable of sequencing up to 700 bases for each reaction. The automated DNA sequencer can simultaneously process up to 100 samples at a time within 3 hours and generate data for 100 unique DNA sequences with about 600-700 bases each. 

Electrophoresis is another separation process that, however, is based on the mobility of ions in an electric field. The different modes of modern capillary electrophoresis with its different separation mechanisms have paid more and more attention during the last decade. 

The method of complete electrolysis is also important in elucidating the mechanism of an electrode reaction. Usually, the substance under study is completely electrolyzed at a controlled potential and the products are identified and determined by appropriate methods, such as gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis. In the GC method, the products are often identified and determined by the standard addition method. If the standard addition method is not applicable, however, other identification/determination techniques such as GC-MS should be used. The HPLC method is convenient when the product is thermally unstable or difficult to vaporize. HPLC instruments equipped with a high-sensitivity UV detector are the most popular, but a more sophisticated system like LC-MS may also be employed. In some cases, the products are separated from the solvent-supporting electrolyte system by such processes as vaporization, extraction and precipitation. If the products need to be collected separately, a preparative chromatographic method is use-... 

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