Conventional electrophoresis instrumentation

As an instrumental approach to conventional electrophoresis, capillary electrophoresis offers the capability of on-line detection, micropreparative operation and automation (6,8,45-47). In addition, the in tandem connection of capillary electrophoresis to other spectroscopy techniques, such as mass spectrometry, provides high information content on many components of the simple or complex peptide under study. For example, it has been possible to separate and characterize various dynorphins by capillary electrophoresis-mass spectrometry (33). Therefore, the combination of CE-mass spectrometry (CE-MS) provides a valuable analytical tool useful for the fast identification and structural characterization of peptides. Recently, it has been demonstrated that the use of atmospheric pressure ionization using Ion Spray Liquid Chromatography/ Mass Spectrometry is well suited for CE/MS (48). This approach to CE/MS provides a very effective and straightforward method which allow the feasibility of obtaining CE/MS data for peptides from actual biological extracts, i.e., analysis of neuropeptides from equine cerebral spinal fluid (33). 

In this section, instrumentation, general electrophoretic operations, technical and practical considerations, and types of conventional electrophoresis are discussed. 

In contrast to the cumbersome and time-consuming tasks of conventional electrophoresis, CE is well suited to automation. Samples are easily applied to the capillary, a variety of detector types can be used, and the resulting electrophore-tograms can be analyzed and manipulated in much the same manner as chromatograms. Commercial instruments resemble many HPLC instruments in terms of automated sample loading and data analysis. Traditional serum protein electrophoresis, for example, can be fully automated with CE. 

Injection valves have been used to introduce samples into a capillary electrophoresis instrument. The injection system is similar to a six-port valve used in a conventional HPLC instrument, where the sample can be loaded into a sample loop on the valve, then the valve actuated to inject the plug onto the capillary. The size of the injection is determined by the plug size and flow rate. This system is not the fastest or most reproducible, but it relies on fairly simple technology. Valved injections have been used in conjunction with separation times down to 10 s. ... 

Capillary electrophoresis employing chiral selectors has been shown to be a useful analytical method to separate enantiomers. Conventionally, instrumental chiral separations have been achieved by gas chromatography and by high performance liquid chromatography.127 In recent years, there has been considerable activity in the separation and characterization of racemic pharmaceuticals by high performance capillary electrophoresis, with particular interest paid to using this technique in modem pharmaceutical analytical laboratories.128 130 The most frequently used chiral selectors in CE are cyclodextrins, crown ethers, chiral surfactants, bile acids, and protein-filled... 

Electrophoresis on cellulose acetate strips (Sepraphore III, Gelman Instrument, Ann Arbor, MI) was done in the conventional manner [12] in order to obtain a comparative electrophoretic mobility of non-adsorbed albumin. For this purpose, BSA-BSA (2.5 w/v) was deposited on the cellulose acetate paper twice in volumes of 10 ul each. Electrophoresis was attain performed in the Gelman Chamber with Pt electrodes at 20°C (see Table 3) After completion, the strips were stained with Ponceau S protein stain (Gelman Instruments) and washed with 5 acetic acid. The stained cellulose acid strips were subsequently cut into 3 mm wide pieces which were monitored for protein content y-count-ing. 

EC detection is a promising alternative for capillary electrophoresis microchips due to its inherent characteristics, allowing a proper miniaturisation of the devices and compatibility with the fabrication processes, in case of an integrated detection. Moreover, the low cost associated permit the employment of disposable elements. As the EC event occurs on the surface of electrodes and the decrease in size usually results in new advantages (see Chapter 32), the possibilities of incorporating EC detectors are broad. The simplicity of the required instrumentation, portable in many cases, suit well with the scaling-down trend. Moreover, as the sample volume in conventional micro-channel devices is less than 1 nL, a very highly sensitive detector should be constructed to analyse even modest concentrations of sample solutions. Since sensitivity is one of the accepted characteristics of EC detection EC-CE microchips approach to the ideal analytical devices. 

Because of polydisperse nature of HS, the importance of separation methods increased as the science evolved. Various separation methods were widely used for conventional fractionation and characterization of components based on differences in component solubility, charge, molecular weight, and/or size, polarity, hydropho-bicity, and so on (Janos, 2003). More recent research focused on advanced molecular-level analyses of humic mixtures (Hertkorn and Schmitt-Kopplin, 2007), in which a combination of separation techniques, mostly, chromatography, or capillary electrophoresis) were coupled with high-resolution instrumental analysis [e.g., mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy]. Several examples appeared in the literature, including those that used size exclusion chro-... 

In traditional analytical chemistry the determination of enantiomeric purity is sometimes carried out by capillary electrophoresis (CE) in which the electrolyte contains chiral selectors such as cyclodextrin (CD) derivatives [54], Unfortunately the conventional form of this analytical technique allows only a few dozen ee determinations per day. However, as a consequence of the analytical demands arising from the Human Genome Project, CE has been revolutionized in recent years so that efficient techniques for instrumental miniaturization are now available, making ultra-high-through-put analysis of biomolecules possible for the first time [55]. Two different approaches have emerged, namely capillary array electrophoresis (CAE) [55a - e] and CE on microchips (also called CAE on chips) [55f - m[. Both techniques can be used to carry out... 

Small drug molecules have not been extensively researched with microchip electrophoresis devices despite the great potential achieved in conventional CE systems. One reason is that most of the small drug molecules possess chromophores thus, UV absorbance detection is the most popular detection mode but it is not universally available for a microchip device such as LIE is because of sensitivity problems. Because of the radiation danger imposed by UV light, proper instrumental setup specific to the microchip format is required. In addition, UV transparent material such as fused silica or fused quartz is required to fabricate the microchip device. Because UV absorbance is much less sensitive than LIE, a special design is necessary to increase the optical path length of the detection flow cell. This has been achieved by some clever approaches pioneered by Harrison and co-workers.Recently, a commercial microchip electrophoresis system with UV detection was released by Shimadzu, which will drive the application of microanalytical techniques to a wide variety of molecules of pharmaceutical interest. 

On the contrary to rod shaped gels, slab gels can be dried on a paper sheet after staining and destaining and stored in this rather conventional form. Commercial instruments for slab gel electrophoresis and the destaining accessories can be obtained from e.g., Pharmacia Fine Chemicals, Uppsala, Sweden Desaga, Heidelberg, FRG or Bio-Rad Laboratories, Richmond, CA, USA. 

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