Other electrophoretic methods

Previous chapters have emphasized the important role of the MWD in determining many functional properties of cereal proteins. Measurement of MWD has met two main problems. The first one is the difficulty of solubilization, particularly of the glutelins. The second is that many of the standard methods for measurement lose their sensitivity above a certain MW. Colligative properties such as osmotic pressure and freezing point depression are related to the number of molecules and therefore give a measure of the number-average MW (MJ. 

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Capillary electrophoresis differs from other electrophoretic methods in two principal ways ... 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Electrophoretic and similar compacted coatings are in early stages of development but will no doubt take their place alongside other coating methods. 

Electrophoretic methods of separation of LD Tsoenzymes have become routine in clinical laboratories. Efforts are now being made to standardize the methodologies used for LD isoenzymes, particularly by Rosalki (38). The preferred methods are based on electrophoresis on a solid medium, so that the several bands may be scanned instrumentally. Differential isoenzyme inhibition with urea or other inhibitors is based on the fact that the heart LD isoenzyme is more resistant to inhibition than other isoenzymes. However, the analyst then has the problem of allocating the observed degree of inhibition between the different isoenzymes of a given sample, a problem that has not been resolved satisfactorily thus far. Hence, differential inhibition is not as reliable for isoenzyme separation as is electrophoresis. 

An important technique for the qualitative and quantitative analysis of different macromolecular materiafs is based on the efectrophoretic separation of particfes having different transport vefocities (e.g., because they have different zeta potentiafs). This technique is used for the anafysis of proteins, pofysaccharides, and other naturally occurring substances whose molecular size approaches that of colloidal particles (for more details, see Section 30.3.4). It is an advantage of the electrophoretic method that mild experimental conditions can be used—dilute solutions with pH values around 7, room temperature, and so on—which are not destructive to the biological macromolecules. 

Other purification methods include a liquid phase chromatography, electrophoretic separation by mass spectroscopy, separation using magnetic properties, and so on. These separation methods are limited only for the metal nanoparticles having a special property useful for these purification methods. 

Modes of Operation There is a close analogy between sedimentation of particles or macromolecules in a gravitational field and their electrophoretic movement in an electric field. Both types of separation have proved valnable not only for analysis of colloids but also for preparative work, at least in the laboratory. Electrophoresis is applicable also for separating mixtures of simple cations or anions in certain cases in which other separating methods are ineffectual. 

As with other chromatographic methods, there are a number of electrophoretic methods, including paper and gel electrophoreses and CE. Electrophoresis uses an electric current to move ionic species, either simple ions, amino acids, or complex proteins, through a medium (i.e., a gel) or a capillary (i.e., CE). During this process, typically, the ionic species move at different rates and are thus separated. 

The zone capillary electrophoretic methods are applied the most and are therefore discussed in more detail. The others are discussed briefly. 

Polyacrylamide gel (PAG) electrophoresis is performed either in cylindrical glass tubes or in flat beds (Figure 3.27). The method is comparable with other electrophoretic techniques but care must be taken to keep the current low to prevent any significant heating effect. 

As the separation characteristics of liquid chromatographic and electrophoretic techniques markedly differ from each other, combined methods using the advantages of both procedures have been successfully used for the analysis of flavonoids. Thus, the use of CZE-UV, HPTLC-UV and GC-MS for the measurement of flavonoids in seeds and root exudates of Lotus pedunculatus has been reported. The rooting solution and seed exudate were passed through cellulose acetate filters to bind the flavonoids. After extraction,... 

An electrophoretic method was developed for the simultaneous determination of artificial sweeteners, preservatives and colours in soft drinks. The samples were degassed by sonication, filtered and used for analysis without any other pretreatment. Measurements were realized in uncoated fused-silica capillaries, the internal diameter being 50 ptm. Capillary lengths were 48.5 cm (40 cm to the detector) and 65.4 cm (56 cm to the detector). Capillaries were conditioned by washing them with (1 M sodium hydroxide (10 min), followed by 0.1 M sodium hydroxide (5 min) and water (5 min). Samples were injected hydrodinamically (250 mbar) at the anodic end. Analyses were performed at a voltage of 20 kV and the capillary temperature was 25°C. Analytes having ionizable substructure... 

During the last few years, a number of specific monographs for different pharmaceutical products have appeared in pharmacopoeias in which CE is prescribed as one of the analytical procedures. Several comparative studies have been reported in which established analytical procedures described in pharmacopoeial monographs were compared with capillary electrophoretic methods or in which CE was evaluated as a valuable additional technique.Based on the results, some analytical procedures have been replaced by CE-based alternative methods in a number of monographs. For other products, CE has been included in the monograph from the initial version onwards. In addition, CE has been applied... 

There are numerous advantages to the FAC approach that differentiate it from many forms of bioassay - MS-dependent or otherwise. The FAC method offers thermodynamic and kinetic binding data from the breakthrough curves. As with the classical application of the FA method, the quality of the data is superb relative to other chromatographic or electrophoretic methods [9, 10]. It is an equilibrium method, as opposed to systems that rely upon the separation of bound from unbound, and this forms the basis of its accuracy. 

In theory, if the net charge, q, on a molecule is known, it should be possible to measure / and obtain information about the hydrodynamic size and shape of that molecule by investigating its mobility in an electric field. Attempts to define /by electrophoresis have not been successful, primarily because Equation 4.3 does not adequately describe the electrophoretic process. Important factors that are not accounted for in the equation are interaction of migrating molecules with the support medium and shielding of the molecules by buffer ions. This means that electrophoresis is not useful for describing specific details about the shape of a molecule. Instead, it has been applied to the analysis of purity and size of macromolecules. Each molecule in a mixture is expected to have a unique charge and size, and its mobility in an electric field will therefore be unique. This expectation forms the basis for analysis and separation by all electrophoretic methods. The technique is especially useful for the analysis of amino acids, peptides, proteins, nucleotides, nucleic acids, and other charged molecules. 

The use of agarose as an electrophoretic method is widespread. The advantages uf agarose electrophoresis are that it requires no additives of cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

From what has been learned over the past year during this assessment phase, The Aerospace Corporation and its subcontractors will be concentrating now on the development of better blood identification methodologies. Improved immunological and electrophoretic methods, as well as combinations of these and other new methods, are being explored for application to the forensic serology problem. Other new blood systems with even higher discrimination capabilities are known but have yet to be adapted for use with dried blood. 

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