Electrophoresis factors affecting

Zhu, M., Hansen, D.L., Burd, S., and Gannon, F., Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis, /. Ckromatogr., 480, 311, 1989. 

Factors Affecting Ionic Migration. Effect of Temperature. pH and Ionic Strength. Electro-osmosis. Supporting Medium. Detection of Separated Components. Applications of Traditional Zone Electrophoresis. High-performance Capillary Electrophoresis. Capillary Electrochromatography. Applications of Capillary El ectrochromatography. ... 

This chapter deals with the validation of capillary electrophoresis (CE) methods. It describes the various validation characteristics, namely accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range in accordance with the official guidelines. Practical aspects related to the calculation of these parameters and factors affecting them in CE analysis have also been described. Validation requirements have been described according to the goal of the method. The chapter contains numerous tables and diagrams to illustrate these ideas. It also covers other related aspects such as instrument qualification, revalidation, and method transfer. 

Capillary electrochromatography (CEC) is a miniaturized separation technique that combines aspects of both interactive chromatography and capillary electrophoresis. In this chapter, the theory of CEC and the factors affecting separation such as the stationary phase and mobile phase parameters have been discussed. The chapter focuses on the types and preparation of columns for CEC and describes the progress made in the development of open-tubular, particle-packed, and monolithic columns. The detection techniques in CEC such as the traditional UV detection and improvements made in coupling with more sensitive detectors such as mass spectrometry are also described. The chapter provides a summary of some applications of CEC in the analysis of pharmaceuticals and biotechnology products. 

IE Valko, HAH Billiet, J Frank, KChAM Luyben. Factors affecting the separation of mandelic acid enantiomers by capillary electrophoresis. Chromatographia 38 730-736, 1994. 

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. 

Another factor affecting particle velocity is the relative contribution of electro-osmotic fluid flow and electrophoresis on the particle. Since the observed velocity is the sum of the intrinsic electrophoretically induced particle velocity and fluid velocity, there exist situations where the two combine to render the particle velocity unobservable within the range discussed above. For similar reasons. there are situations where particle velocity may be observable outside the range discussed above. Choice of particle is important in being able to extract the electrokinetic information. 

Factors affecting ionic migration. F.ffecl of temperature. pH ami ionic strength. Elcciroosmosis. Supporting medium. Detection oF.separated components. Applications of tradional zone electrophoresis. High per- formance capillary electrophoresis. 

J. Lunney, A. Chrambach, and D. Rodbard, Anal. Biochem., 40 158-173 (1971). Factors Affecting Resolution, Bandwidth, Number of Theoretical Plates and Apparent Diffusion Coefficients in Polyacrylamide Gel Electrophoresis. 

Table 10,5 Factors affecting capillary electrophoresis separations...

A. Weston, P. Brown, E Jandik, W. R. Jones and A. L. Heckenberg, Factors affecting the separation of inorganic metal cations by capillary electrophoresis, /. Chromatogr.. 593,289,1992. 

A. D Emanuele and J. N. Staniforth, An electrically modulated (hug delivery device. III. Factors affecting drug stability during electrophoresis, Pharm. Res. 9, 312-315(1992). 

Tsuji, K. Factors affecting the performance of SDS gel-filled capillary electrophoresis. J. Chromatogr. A, 661, 257, 1994b. 

Wan H., Schmidt S., Carlsson L., Blomberg L.G, Some factors affecting enantiomeric impurity determination by capillary electrophoresis using ultraviolet and laser-induced fluorescence detection. Electrophoresis, 20, 2705-2714 (1999). 

Voltages used fall in the range of 10-30 kV. This translates in currents in the range of 20-100 mA. Higher currents are avoided usually because they produce too much heat inside the capillary (see "Factors Affecting Electrophoresis" discussed earlier in this chapter). This can result in peak broadening and loss of resolution. 

After electrophoresis, protein bands are transferred onto a solid support. Many aspects of a transfer can affect antigen detection. Some of these factors are specific to the transfer method and choice of membrane, whereas others apply to the entire blotting procedure. For example, the transfer efficiency may be affected by the presence of SDS in the gel and whether the gel was stained prior to transfer. 

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