Electrophoretic mobility factors affecting

In the following, we will take a brief look at additional factors that affect the electrophoretic mobility and thus separation of charged particles. 

Figure 12.12b illustrates the application of gel electrophoresis to protein characterization. In this illustration a cross-linked polyacrylamide gel is the site of the electrophoretic migration of proteins that have been treated with sodium dodecyl sulfate. The surfactant dissociates the protein molecules into their constituent polypeptide chains. The results shown in Figure 12.12b were determined with well-characterized polypeptide standards and serve as a calibration curve in terms of which the mobility of an unknown may be interpreted to yield the molecular weight of the protein. As with any experiment that relies on prior calibration, the successful application of this method requires that the unknown and the standard be treated in the same way. This includes such considerations as the degree of cross-linking in the gel, the pH of the medium, and the sodium dodecyl sulfate concentration. The last two factors affect the charge of the protein molecules by dissociation and adsorption, respectively. Example 12.5 considers a similar application of electrophoresis. 

There are a number of different factors which may affect the level of uptake and the energetics of adsorption from solution the chemistry and electrical properties of the solid surface and the molecular/micellar/polymeric structure of the solution must all be taken into account. Whenever possible, a study of both adsorption isotherms and enthalpies of displacement is worthwhile, but it is often necessary to complement these measurements with others including electrophoretic mobilities, FI7R spectra-and various types of microscopy. 

Figure 13. Electrophoretic mobility (Fen Kem 3000) of the emulsion from Figure 12 after cationic polymer addition (A). The cationic polymer has neutralized the oil droplet surface charge and electrostatically destabilized the emulsion. The photomicrograph (B) shows this destabilized emulsion that has begun to flocculate or a lomerate but that is not coalescing. This electrostatic destabilization is not the only factor affecting emulsion stability. Factors such as interfacial tension and film strength can prevent coalescence of the emulsion droplets, even though they can now closely approach each other and ag omer-...

The velocity of a solute in the capillary is determined by its electrophoretic mobility and electro-osmotic flow (EOF), which are affected by temperature, and which is influenced by the diameter and length of the capillary, its contents, concentration, and pH of running buffer, applied voltage, current, viscosity, and zeta-potential. The subtle variation of EOF is also a main factor in maintaining high reproducibility if CE is automated by the constant temperature of the capillary and running buffer with proper buffering capacity. However, it is difficult to keep the temperature constant, as EOF depends on the condition of the fused silica. 

A number of factors influence the separation which can be achieved. The shape of the molecule and its polarity determine whether it will be able to enter the hydrophobic cavity of the cyclodextrin. Vt varying the concentration of cyclodextrin, one can influence the partition of the complex between the selective and bulk phase. One can also modify the cyclodextrin in a manner that changes its solubility or affects its electrophoretic mobility. Bile salts and crown ethers have also been used as enantioselective agents. 

The major limitation of PALS is that no mobility distribution information can be obtained and the type of mean is not defined. In addition, the accuracy of the measured electrophoretic mobility depends on the accuracy of the scattering vector K, which can te determined quite accurately based on instrument setup, and the accuracy of A, which is often affected by experimental noise and is difficult to ascertain. Other factors, such as electroosmosis, electronic artifact and the choice of a correct field frequency can also affect the measurement accuracy, as demonstrated in Figure 6.31, in which correct electrophoretic mobihty value using PALS can only be obtained at electric field frequencies between 30-200 Hz. 

Method validation includes determination of performance characteristics such as selectivity (which determines accuracy), linearity, precision, and sensitivity (limit of detection). This work evaluated linearity, precision, and sensitivity for specific CZE separation conditions selectivity was reported previously (15). Factors that contribute to assay imprecision by affecting peak shape (such as the pH of the mobile phase) or migration velocity (pH effects on the electrophoretic velocity) were evaluated also. 

The peptides, which are composed of only a few amino acids, exhibit predictable behavior in CZE in that their mobility (electrophoretic migration) can be predicted on the basis of their size (mass) and their charge characteristics. The charge of such peptides can be predicted from the pKa values of individual amino acids contained in them. For larger peptides and proteins, the calculation of charge based on ionization constant is not trivial and can not be easily calculated based on the pKas of the free amino aids. Besides the mass-to-charge ratio, the other factors that affect mobility are hydrophobic-ity, primary sequence, conformational difference, and the chirality of amino acids. This point has been demonstrated by an investigation on several nonapeptides of identical composition but with different primary sequences ... 

Another factor and potential problem that can affect mobility is wick flow. During electrophoresis heat evolved because of the passage of current through a resistive medium can cause evaporation of solvent from the electrophoretic support. This drying effect draws buffer into the support fi om both buffer compartments. If significant, the flow of buffer from both directions can affect protein migration and hence the calculated mobility. 

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