Capillary electrophoresis detection techniques

The rapid separations offered by capillary electrophoresis have made it amenable as a detector in hyphenated techniques. For LC-CE, the total analysis time is usually governed by the LC separation, which generally takes minutes. However, capillary electrophoresis detection adds more peak capacity because of a second and orthogonal dimension for separation, and shorter separation conditions for LC can often be tolerated. For example, a 2.5 min reversed-phase liquid chromatography gradient was used in conjunction with 2.5 s CE separations for the detection of a tryptic digest of cytochrome c. ... 

Capillary electrophoresis (CE) techniques have been used for selective orthophosphate analysis. CE separations of anions in waters are much faster than ion chromatography, but detection of phosphate by UV absorbance is very insensitive. This may be overcome by on-capillary preconcentration using isotacho-phoresis, which enables sub-pgl detection limits to be achieved in high ionic strength matrices. 

Luminol-based chemiluminescence methods have also been employed for detection of analytes in flowing stream analytical techniques such capillary electrophoresis (282), flow injection analyses, and hplc (267). AppHcations of the enhanced luminol methodology to replace radioassay methods have been developed for a number of immunological labeling techniques (121,283). 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

The spectrum of new analytical techniques includes superior separation techniques and sophisticated detection methods. Most of the novel instruments are hyphenated, where the separation and detection elements are combined, allowing efficient use of materials sometimes available only in minute quantities. The hyphenated techniques also significantly increase the information content of the analysis. Recent developments in separation sciences are directed towards micro-analytical techniques, including capillary gas chromatography, microbore high performance liquid chromatography, and capillary electrophoresis. 

A number of developments have increased the importance of capillary electrophoretic methods relative to pumped column methods in analysis. Interactions of analytes with the capillary wall are better understood, inspiring the development of means to minimize wall effects. Capillary electrophoresis (CE) has been standardized to the point of being useful as a routine technique. Incremental improvements in column coating techniques, buffer preparation, and injection techniques, combined with substantive advances in miniaturization and detection have potentiated rugged operation and high capacity massive parallelism in analysis. 

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. 

Schure, M.R. (1999). Limit of detection, dilution factors, and technique compatibility in multidimensional chromatography, capillary electrophoresis, and field-flow fractionation. Anal. Chem. 71, 1645-1657. 

Identification and quantification of natural dyes need high performance analytical techniques, appropriate for the analysis of materials of complicated matrices containing a small amount of coloured substances. This requirement perfectly fits coupling of modern separation modules (usually high performance liquid chromatography in reversed phase mode, RPLC, but also capillary electrophoresis, CE) with selective detection units (mainly mass spectrometer). 

In the 1990s, CL detection was coupled as a detection system in the by then recently introduced separation technique in routine analysis, capillary electrophoresis (CE). Hara s group reported the first application in 1991 [234], using... 

CL reactions can be coupled as a detection technique in chromatography, capillary electrophoresis, or immunoassay, providing qualitative and/or quantitative information of a large variety of species in the gas and liquid phases. 

Also, specific chapters deal with the use of CL reactions as detection mode in FIA (Chapter 12), in separational techniques, such as liquid chromatography (LC) (Chapter 14) or capillary electrophoresis (CE) (Chapter 15), in immunoassay (Chapter 18), and in the development of sensors (Chapter 20). The recent use of this technique for the analysis of DNA (Chapter 19) and a photosensitized CL mode for medical routine and industrial applications (Chapter 17) are also considered in this book. 

As can be seen in Table 3, a wide range of analytes derivatized with different labels have been detected using the POCL reaction. Most of these applications have employed flow injection or liquid chromatographic techniques. An area of growing interest is the combination of capillary electrophoresis with chemiluminescence. Several strategies have been used to detect analytes with fluorescent... 

In analytical chemistry there is an ever-increasing demand for rapid, sensitive, low-cost, and selective detection methods. When POCL has been employed as a detection method in combination with separation techniques, it has been shown to meet many of these requirements. Since 1977, when the first application dealing with detection of fluorophores was published [60], numerous articles have appeared in the literature [6-8], However, significant problems are still encountered with derivatization reactions, as outlined earlier. Consequently, improvements in the efficiency of labeling reactions will ultimately lead to significant improvements in the detection of these analytes by the POCL reaction. A promising trend is to apply this sensitive chemistry in other techniques, e.g., in supercritical fluid chromatography [186] and capillary electrophoresis [56-59], An alter-... 

This section deals briefly with classical methods based on conventional mixing of the sample and reagents such as the batch mode and low-pressure flow mixing methods, as well as the use of CL detection in continuous separation techniques such as liquid chromatography and capillary electrophoresis for comparison with the unconventional mixing mode. 

Optimization and applications of CL detection in flow injection and liquid chromatographic analysis and the relatively new use of CL in capillary electrophoresis are extensively described. Particular interest is attached to the universally applied peroxyoxalate CL reactions, as well as to the applications of new acridan esters in immunoassay. Obviously, the related applications of BL and CL imaging techniques in analytical chemistry, and the increasing importance of these techniques in DNA analysis—including the recent strategies in the development of CL sensors—are also presented. 

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